Humanized anti-OX40 antibodies and uses thereof

ABSTRACT

The disclosure provides humanized anti-OX40 antibodies. Also provided are methods of making such antibodies, and methods of use, e.g., treatment of cancer.

CROSS REFERENCE TO RELATED APPLICATIONS

This claims benefit of U.S. Provisional Application No. 62/062,431 filedOct. 10, 2014. The above listed application is incorporated by referenceherein in its entirety for all purposes.

REFERENCE TO THE SEQUENCE LISTING

This application incorporates by reference a Sequence Listing submittedwith this application as text file entitled OX40H-100US1_SL.txt createdon Oct. 6, 2015 and having a size of 865 kilobytes.

BACKGROUND

OX40 (CD134; TNFRSF4) is a tumor necrosis factor receptor foundprimarily on activated CD4⁺ and CD8⁺ T cells, regulatory T (Treg) cellsand natural killer (NK) cells (Croft et al., 2009, Immunol Rev.229:173-91). OX40 has one known endogenous ligand, OX40 ligand (OX40L;CD152; TNFSF4), which exists in a trimeric form and can cluster OX40,resulting in potent cell signaling events within T cells. Id. Signalingthrough OX40 on activated CD4⁺ and CD8⁺ T cells leads to enhancedcytokine production, granzyme and perforin release, and expansion ofeffector and memory T cell pools (Jensen et al., 2010, Semin Oncol.37:524-32). In addition, OX40 signaling on Treg cells inhibits expansionof Tregs, shuts down the induction of Tregs and blocks Treg-suppressivefunction (Voo et al., 2013, J Immunol. 191:3641-50; Vu et al., 2007,Blood. 110:2501-10).

Immunohistochemistry studies and early flow cytometry analyses showedthat OX40 is expressed on T cells infiltrating a broad range of humancancers (Baruah et al., 2011, Immunobiology 217:668-675; Curti et al,2013, Cancer Res. 73:7189-98; Ladanyi et al, 2004, Clin Cancer Res.10:521-30; Petty et al, 2002, Am J Surg. 183:512-8; Ramstad et al, 2000,Am J Surg. 179:400-6; Sarff et al, 2008, Am J Surg. 195:621-5;discussion 625; Vetto et al, 1997, Am J Surg. 174:258-65). While notwishing to be bound by theory, OX40 expression on tumor-infiltratinglymphocytes correlates with longer survival in several human cancers,suggesting that OX40 signals can play a role in establishing anantitumor immune response (Ladanyi et al., 2004, Clin Cancer Res.10:521-30; Petty et al., 2002, Am J Surg. 183:512-8).

In a variety of nonclinical mouse tumor models, agonists of OX40,including antibodies and OX40 ligand fusion proteins, have been usedsuccessfully with promising results (Kjaergaard et al., 2000, CancerRes. 60:5514-21; Ndhlovu et al., 2001, J Immunol. 167:2991-9; Weinberget al., 2000, J Immunol. 164:2160-9). Co-stimulating T cells throughOX40 promoted anti-tumor activity that in some cases was durable,providing long-lasting protection against subsequent tumor challenge(Weinberg et al., 2000, J Immunol. 164:2160-9). Treg-cell inhibition andco-stimulation of effector T cells were shown to be necessary for tumorgrowth inhibition of OX40 agonists (Piconese et al., 2008, J Exp Med.205:825-39). Many strategies and technologies have been explored toenhance the anti-tumor effect of OX40 agonist therapy throughcombinations with vaccines, chemotherapy, radiotherapy, andimmunotherapy (Jensen et al., 2010, Semin Oncol. 37:524-32; Melero etal., 2013, Clin Cancer Res. 19:997-1008).

SUMMARY

This disclosure provides antibodies that bind to OX40, e.g., human OX40.In certain aspects the antibodies provided are humanized antibodies. Forexample, this disclosure provides an antibody or antigen-bindingfragment thereof that includes a humanized heavy chain variable region(VH) and a humanized light chain variable region (VL), where the VHincludes an amino acid sequence with the formula:HFW1-HCDR1-HFW2-HCDR2-HFW3-HCDR3-HFW4,where HFW1 is SEQ ID NO: 6 or SEQ ID NO: 7, HCDR1 is SEQ ID NO: 8, HFW2is SEQ ID NO: 9 (WIRX₃₉HPGKGLEX₄₇X₄₈G; where X₃₉ is Q or K, X₄₇ is W orY, and X₄₈ is I or M), HCDR2 is SEQ ID NO: 14, SEQ ID NO: 15 or SEQ IDNO: 16, HFW3 is SEQ ID NO: 17 (RITINX₇₁DTSKNQX₇₈SLQLNSVTPEDTAVYX₉₁CAR;where X₇₁ is P or R, X₇₈ is F or Y, and X₉₁ is Y or F, HCDR3 is SEQ IDNO: 25, SEQ ID NO: 26, or SEQ ID NO: 27), and HFW4 is SEQ ID NO: 28,where the VL includes the amino acid sequence SEQ ID NO: 29 or SEQ IDNO: 32; and where the antibody or fragment thereof can specifically bindto human OX40. In certain aspects, the amino acid sequence of HFW2 isSEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 1, and incertain aspects the the amino acid sequence of HFW3 is SEQ ID NO: 18,SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO:23, or SEQ ID NO: 24.

In certain aspects, the VH of the provided antibody or fragment thereofincludes the amino acid sequence SEQ ID NO: 33, SEQ ID NO: 35, SEQ IDNO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55,SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO:65, or SEQ ID NO: 67. In certain aspects, the VL of the providedantibody or fragment thereof includes the amino acid sequence SEQ ID NO:29 and the VH includes the amino acid sequence SEQ ID NO: 59.

In certain aspects, the provided antibody or fragment thereof furtherincludes a light chain constant region or fragment thereof fused to theC-terminus of the VL, e.g., a human kappa constant region or a humanlambda constant region. In certain aspects, the provided antibody orfragment thereof further includes a heavy chain constant region orfragment thereof fused to the C-terminus of the VH, e.g., a human IgG1constant region, a human IgG4P constant region, a human IgG1TM constantregion or a murine IgG1 constant region. In certain aspects the heavychain constant region is a human IgG1 constant region. In certainaspects, the provided antibody or fragment thereof includes the heavychain amino acid sequence SEQ ID NO: 71 and the light chain amino acidsequence SEQ ID NO: 30. An antigen-binding fragment of an antibody asprovided by this disclosure can be, e.g., an Fv fragment, an Fabfragment, an F(ab′)2 fragment, an Fab′ fragment, a dsFv fragment, anscFv fragment, or an sc(Fv)2 fragment, or any combination thereof.

In certain aspects, the provided antibody or fragment thereof canspecifically bind to human, cynomolgus monkey, or rhesus monkey OX40,e.g., can specifically bind to OX40 as expressed on Jurkat cells,primary activated CD4⁺ or CD8⁺ T cells from human, cynomolgus monkey,rhesus monkey, or any combination thereof. In certain aspects, theprovided antibody or fragment thereof does not bind to murine or ratOX40. In certain aspects, the provided antibody or fragment thereof doesnot cross react with related TNFRSF proteins.

In certain aspects, the provided antibody or fragment thereof can have abinding affinity for human OX40 expressed on primary activated humanCD4⁺ T cells of about 250 pM to about 370 pM as measured by flowcytometry, e.g., about 312 pM. In certain aspects, the provided antibodyor fragment thereof can achieve 20% receptor occupancy on primaryactivated human CD4⁺ T cells (EC₂₀) at about 63 to about 93 pM, 50%receptor occupancy on primary activated human CD4⁺ T cells (EC₅₀) atabout 250 to about 370 pM, and 90% receptor occupancy on primaryactivated human CD4⁺ T cells (EC₉₀) at about 2290 to about 3330 pM asmeasured by flow cytometry. For example, in certain aspects, EC₂₀ isabout 78 pM, EC₅₀ is about 312 pM, and EC₉₀ is about 2810 pM.

In certain aspects, the provided antibody or fragment thereof can have abinding affinity for human OX40 expressed on OX40-overexpressing Jurkatcells of about 250 pM to about 600 pM as measured by flow cytometry,e.g., about 424 pM. In certain aspects, the provided antibody orfragment thereof can achieve EC₂₀ on OX40-overexpressing Jurkat cells atabout 60 to about 150 pM, EC₅₀ on OX40-overexpressing Jurkat cells atabout 250 to about 600 pM, and EC₉₀ on OX40-overexpressing Jurkat cellsat about 2260 to about 4380 pM as measured by flow cytometry. Forexample, in certain aspects, EC₂₀ is about 106 pM, EC₅₀ is about 424 pM,and EC₉₀ is about 3820 pM.

In certain aspects, the provided antibody or fragment thereof can have abinding affinity for cynomolgus monkey OX40 expressed on primaryactivated cynomolgus monkey CD4⁺ T cells of about 340 pM to about 820 pMas measured by flow cytometry, e.g., about 580 pM. In certain aspects,the provided antibody or fragment thereof can have a binding affinityfor rhesus monkey OX40 expressed on primary activated rhesus monkey CD4⁺T cells of about 130 pM to about 600 pM as measured by flow cytometry,e.g., about 370 pM.

In certain aspects, the provided antibody or fragment thereof can inducedose-dependent proliferation of activated CD4⁺ T cells anddose-dependent cytokine release in primary activated CD4⁺ T cells in aplate-based assay. For example, in certain aspects, a 20% maximalproliferation response (EC₂₀) can be achieved in primary activated humanCD4⁺ T cells at an antibody concentration of about 14 pM to about 28 pM,a 50% maximal proliferation response (EC₅₀) can be achieved in primaryactivated human CD4⁺ T cells at an antibody concentration of about 0.3pM to about 130 pM, and a 90% maximal proliferation response (EC₉₀) canbe achieved in primary activated human CD4⁺ T cells at an antibodyconcentration of about 50 pM to about 90 pM, all as measured by flowcytometry. In certain aspects, EC₂₀ is about 21 pM, EC₅₀ is about 28 pM,and EC₉₀ is about 72 pM. The released cytokine in primary activatedhuman CD4⁺ T cells can be one, two, three or more of, withoutlimitation, IFNγ, TNFα, IL-5, IL-10, IL-2, IL-4, IL-13, IL-8, IL-12 p70,IL-1β, or any combination thereof, for example, IFNγ, TNFα, IL-5, IL-10,IL-13, or any combination thereof. In certain aspects, the providedantibody or fragment thereof can achieve CD4⁺ T cell proliferation andcytokine release in primary activated cynomolgus monkey CD4⁺ T cells andin primary activated rhesus monkey CD4⁺ T cells.

In certain aspects, the provided antibody or fragment thereof canactivate the NFκB pathway in OX40 expressing T cells in the presence ofFcγR-expressing cells. For example, the OX40-expressing T cells can beOX40-overexpressing Jurkat NFκB-luciferase reporter cells that produceluciferase in response to stimulation of the NFκB signaling pathway. Incertain aspects, the provided antibody or fragment thereof can triggercomplement-dependent or antibody-dependent cellular cytotoxicity againstOX40-expressing cells. In certain aspects, the provided antibody orfragment thereof can bind to human C1q and trigger NK-mediatedantibody-dependent cellular cytotoxicity against the OX40-expressingcells.

In certain aspects, administration of an effective dose of the providedantibody or fragment thereof to a subject in need of cancer treatmentcan inhibit tumor growth in the subject. For example, the tumor growthinhibition can be achieved in the presence of T cells. In certainaspects, tumor growth is inhibited by at least 10%, at least 20%, atleast 30%, at least 40%, and least 50%, at least 60%, or at least 70%compared to administration of an isotype-matched control antibody orfragment thereof.

This disclosure further provides a composition including the antibody orfragment thereof as described above, and a carrier.

This disclosure further provides a polynucleotide that includes anucleic acid that encodes the provided antibody or fragment thereof, orencodes a polypeptide subunit of the provided antibody or fragmentthereof. In certain aspects, the provided polynucleotide includes thenucleic acid of SEQ ID NO: 60, the nucleic acid of SEQ ID NO: 31, thenucleic acid of SEQ ID NO: 72, or any combination thereof. Thisdisclosure further provides a vector that includes the providedpolynucleotide and a host cell that includes the provided polynucleotideor the provided vector. In another aspect, the disclosure provides amethod of producing an antibody or fragment thereof, where the methodincludes culturing the provided host cell under conditions in which theantibody or fragment thereof encoded by the polynucleotide is expressed,and recovering the antibody or fragment thereof.

In additional aspects, the disclosure provides a method to promotesurvival or proliferation of activated T cells, where the methodincludes contacting activated T cells with the provided antibody orfragment thereof, and where the antibody or fragment thereof canspecifically bind to OX40 on the surface of the T cells.

In additional aspects, the disclosure provides a method of inducingcytokine release from activated T cells, where the method includescontacting activated T cells with the provided antibody or fragmentthereof, and where the antibody or fragment thereof can specificallybind to OX40 on the surface of the T cells. In certain aspects thereleased cytokine can be one, two, three or more of, without limitation,IFNγ, TNFα, IL-5, IL-10, IL-2, IL-4, IL-13, IL-8, IL-12 p70, IL-1θ , orany combination thereof, e.g., IFNγ, TNFα, IL-5, IL-10, IL-13, or anycombination thereof. In certain aspects, the activated T cells areactivated CD4⁺ T cells, activated CD8⁺ T cells, or a combinationthereof. In certain aspects, the activated CD4⁺ T cells are human CD4⁺ Tcells, cynomolgus monkey CD4⁺ T cells, rhesus monkey CD4⁺ T cells, or acombination thereof.

In additional aspects, the disclosure provides a method of promoting Tcell activation, where the method includes contacting T cells with theprovided antibody or fragment thereof, where the antibody or fragmentthereof can specifically bind to OX40 on the surface of the T cells. Incertain aspects, T cell activation can be measured through stimulationof the NFκB signal transduction pathway. In certain aspects, the T cellsare activated CD4⁺ T cells, activated CD8⁺ T cells, or a combinationthereof. In certain aspects, the activated CD4⁺ T cells are human CD4⁺ Tcells, cynomolgus monkey CD4⁺ T cells, rhesus monkey CD4⁺ T cells, or acombination thereof. In certain aspects, the contacting includesadministering an effective amount of the antibody or fragment thereof toa subject.

In additional aspects, the disclosure provides a method of treatingcancer in a subject, where the method includes administering to asubject in need of treatment an effective amount of the providedantibody or fragment thereof, or the provided composition. In certainaspects, the cancer is a solid tumor. In certain aspects, administrationof the antibody or fragment thereof or composition can inhibit tumorgrowth, can promote tumor reduction, or both. In certain aspects, tumorgrowth inhibition is achieved in the presence of T cells.

In additional aspects, the disclosure provides a method of enhancing animmune response in a subject, where the method includes administering toa subject in need thereof a therapeutically effective amount of theprovided antibody or fragment thereof, or the provided composition.

In the therapeutic methods provided by this disclosure, the subject tobe treated can be a human subject.

In certain aspects, the provided antibody or fragment thereof can bindto an epitope of human OX40 that falls within amino acids 108 to 146 ofSEQ ID NO: 91. In certain aspects, the epitope includes at least aminoacids leucine 116 (L116) and alanine 126 (A126) of SEQ ID NO: 91. Incertain aspects, the provided antibody or fragment thereof can bind to amouse OX40 variant that has the amino acid sequence of SEQ ID NO: 92,except for a Q113L mutation and a V124A mutation.

This disclosure further provides an isolated peptide consisting of 100or fewer amino acids, where the peptide can be specifically bound by theprovided antibody or fragment thereof. In certain aspects, the peptideincludes amino acids 116 to 126 of SEQ ID NO: 91. In certain aspects,the peptide includes amino acids 108 to 146 of SEQ ID NO: 91 except forone, two, three, four, five, or six single amino acid substitutions,deletions, or insertions at any position except L116 and A126.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1. Mutations in humanized 9B12 VH regions: Numbers in the clonalnick name represent the position of the amino acid in VH, according toKabat numbering. Mabs 1, 2, 5 and 8 are VH chimeric variants paired withhumanized VL. Mabs 10-17 are humanized VH variants with mouse backmutations. Mabs 18-27 are variants engineered to remove potentialsequence liabilities. Variable regions of Mab24 and mAb27 were graftedonto different heavy constant regions for the resulting isotype variantsnamed mAb28-30 and mAb31-32, mAb37, respectively.

FIGS. 2A-D. Binding of OX40mAb24 and 9B12 to OX40 Expressed on theSurface of Primary Activated Human CD4⁺ T cells. MFI=mean fluorescenceintensity of AlexaFluor® 647 labeled secondary anti-human antibodybinding to OX40mAb24 (2A-B) or AlexaFluor® 488 labeled anti-mousesecondary antibody binding to 9B12 (2C-D) on primary human CD4⁺ T cells.

FIGS. 3A-F. Binding of OX40mAb24 and 9B12 to Human OX40 Expressed onJurkat T cells. MFI=mean fluorescence intensity of AlexaFluor® 647labeled secondary anti-human antibody binding to OX40mAb24 (3A-C) orAlexaFluor® 488 labeled anti-mouse secondary antibody binding to 9B12(3D-F) on Jurkat T cells.

FIGS. 4A-B. Binding of OX40mAb24 to TNFRSF-expressing HEK293 cells andOX40-expressing Jurkat T cells. (4A) Transient expression of TNFRSFmembers, as indicated to left of histograms, in HEK293 cells, andbinding to TNFRSF-specific mAbs or to OX40mAb24, as indicated abovehistograms. Gray histogram, fluorochrome-conjugated isotype controlantibody binding for TNFRSF-specific mAb, or goat anti-human AlexaFluor®647 secondary antibody binding control for OX40mAb24; Open histogram,TNFRSF-specific mAb or OX40mAb24 binding. (4B): Binding of OX40mAb24 toOX40-expressing Jurkat as a positive control. Gray histogram, goatanti-human AlexaFluor® 647 secondary antibody binding control; Openhistogram, OX40mAb24 binding.

FIGS. 5A-B. Binding of OX40mAb24 (OX40mAb29)(complementarity-determining regions) CDRs and 9B12 to recombinant humanTNFRSF members by ELISA. Results of ELISA assays demonstrating specificbinding of OX40 by OX40mAb29 (5A) and 9B12 (5B). OX40mAb29 contains theCDRs of OX40mAb24. Antibody binding of OX40 was compared to binding ofother human TNFRSF proteins.

FIG. 6. Schematic diagram of the OX40mAb24 plate-bound bioactivityassay. Q=OX40mAb24; Y=anti-human CD3 antibody clone OKT3.

FIGS. 7A-C. Human CD4⁺ T cell Proliferation in response to OX40mAb24 and9B12. (7A) CD4 T cell proliferation of four independent donors mediatedby plate-immobilized OX40mAb24 in combination with sub-mitogenic TCRstimulation (anti-CD3). Data points were normalized to the lowerasymptotic value of raw data curves prior to graphing to enhancevisualization of the dynamic range of each response. (7B) Representativeraw data from Donor 651 demonstrating proliferation-driven by OX40mAb24plus sub-mitogenic TCR stimulation (anti-CD3) and the relative lack ofproliferation mediated by the R347 human IgG1 control mAb, solubleOX40mAb24 in the presence or absence of concomitant TCR signaling,anti-CD3 mAb alone without OX40mAb24, and by plate-immobilized OX40mAb24in the absence of anti-CD3 mAb. (7C) CD4 T cell proliferation of fourindependent donors mediated by plate-immobilized 9B12 in combinationwith sub-mitogenic TCR stimulation. Symbols, mean values; Error bars,standard deviation of the mean; n=3 technical replicates for OX40mAb24,R347 human IgG1 control mAb, and 9B12 all in combination with anti-CD3;n=2 technical replicates for soluble OX40mAb24, and CD3 in the absenceof OX40mAb24, plate bound OX40mAb24 with no anti-CD3, and solubleOX40mAb24 with no anti-CD3.

FIGS. 8A-E. Human CD4⁺ T cell Cytokine Release in Response to OX40mAb24.Representative OX40mAb24 induced human CD4 T cell cytokine release forDonor 651, including (8A) IFNγ, (8B) TNFα (8C) IL-10 (8D) IL-13, and(8E) IL-5. Symbols, mean values; Error bars, standard deviation of themean; n=3 technical replicates for OX40mAb24 and R347 human IgG1 controlmAb, both in combination with anti-CD3; n=2 technical replicates forsoluble OX40mAb24, soluble OX40mAb24 with no anti-CD3, and anti-CD3 inthe absence of OX40mAb24.

FIGS. 9A-E. Human CD4⁺ T cell Cytokine Release in Response to 9B12.Representative 9B12 induced human CD4 T cell cytokine release for Donor651, including (9A) IFNγ, (9B) TNFα (9C) IL-10 (9D) IL-13, and (9E)IL-5. Symbols, mean values; Error bars, standard deviation of the mean;n=3 technical replicates for 9B12 and mouse IgG1 control mAb, both incombination with anti-CD3; n=2 technical replicates for soluble 9B12,soluble 9B12 with no anti-CD3, and anti-CD3 in the absence of 9B12.

FIG. 10 is a schematic illustrating cell systems used for measuringOX40mAb24 and 9B12 bioactivity. OX40mAb24 cross linking byFcγR-expressing cells mediates the clustering and activation of OX40 onthe cell surface of an OX40-expressing Jurkat NFκB-luciferase reportercell line, resulting in the NFκB-mediated production of luciferase thatcan be measured as a surrogate for OX40 activation. FcγR=fragmentcrystallizable gamma receptor; NFκB=nuclear factor kappa B.

FIGS. 11A-D. Bioactivity of OX40mAb24 in OX40-expressing Jurkat NFκBReporter Cells With and Without Cross-linking by FcγR.Concentration-dependent activity (in RLU) of OX40mAb24-induced signalingthrough human OX40 expressed on the cell surface of a JurkatNFκB-luciferase reporter cell line in a 2-cell bioassay. Reporteractivity after cross-linking of OX40mAb24 by (11A) CD32A-expressingHEK293 cells, and HEK293 parental cells (HEK), or in the presence of(11B) CD32B-expressing HEK293 cells, (11C) Raji B cells, or (11D) CD45⁺cells isolated from a primary lung tumor. Data is representative ofother results found in Table 5-2. mAb=monoclonal antibody, RLU=relativelight units.

FIGS. 12A-D. Bioactivity of 9B12 in OX40-expressing Jurkat NFκB ReporterCells using FcγR Cross-linking by Different Cell Types in 2-CellBioactivity Assays. Concentration-dependent activity (in RLU) of 9B12induced signaling through human OX40 expressed on the cell surface of aJurkat NFκB-luciferase reporter cell lines in a two-cell bioassay.Reporter activity after cross-linking of 9B12 by (12A) CD32A-expressingHEK293 cells, (12B) CD32B-expressing HEK293 cells, (12C) Raji B cells,or (12D) CD45⁺ cells isolated from a primary lung tumor. Data isrepresentative of other results found in Table 5-3. mAb=monoclonalantibody; RLU=relative light units.

FIGS. 13A-B. Natural Killer Cell-mediated Antibody-Dependent CellularCytotoxicity of OX40mAb24, Experiment 1. (13A) Specific killing ofOX40-expressing activated CD4⁺ T cells by human NK cells from anallogeneic, left, or autologous, right, NK and CD4⁺ T cell donor pairsusing 10 μ/mL of 9B12, OX40mAb24, or the IgG1 triple mutant (mAb29) orhuman IgG4P (mAb28) versions of OX40mAb24. (13B) Lysis of Toledo B cellsby NK cells from donors 350 and 351 in the presence of rituximab, butnot R347 human IgG1 isotype control antibody. Technical replicates wereconducted in triplicate. Error bars represent standard error of themean. ADCC=antibody-drug-dependent-cytotoxicity; mAb=monoclonalantibody; NK=natural killer.

FIGS. 14A-B. Natural Killer Cell-mediated Antibody-Dependent CellularCytotoxicity of OX40mAb24, Experiment 2. (14A) Specific killing ofOX40-expressing activated CD4 T cells by human NK cells from anallogeneic, left, or autologous, right, NK and CD4 T cell donor pairsusing 10 μ/mL of control R347 human IgG1, 9B12, OX40mAb24, the humanIgG4P (mAb28) or the IgG1 triple mutant (mAb29) versions of OX40mAb24.(14B) Lysis of Toledo B cells by NK cells from donors 558 and 589 in thepresence of rituximab, but not R347 human IgG1 isotype control antibody.Technical replicates were conducted in triplicate. Error bars representstandard error of the mean. ADCC=antibody-drug-dependent-cytotoxicity;mAb=monoclonal antibody; NK=natural killer.

FIGS. 15A-D. Assessment of Natural Killer Cell-mediatedAntibody-Dependent Cellular Cytotoxicity of OX40mAb24 and 9B12,Experiment 3. (15A-B) Specific killing of OX40-expressing human CD4 Tcells mediated by OX40mAb24, but not by 9B12, using primary human NKcells from two separate donors as indicated. (15C-D) Lysis of Toledo Bcells by NK cells from donors 363 and 504 in the presence of rituximab,but not R347 human IgG1 isotype control antibody. Technical replicateswere conducted in duplicate. Error bars represent standard error of themean. ADCC=antibody-drug-dependent-cytotoxicity; mAb=monoclonalantibody; NK=natural killer.

FIGS. 16A-D. Assessment of Natural Killer Cell-mediatedAntibody-Dependent Cellular Cytotoxicity of OX40mAb24, Experiment 4.(16A-B) Specific killing of OX40-expressing human CD4 T cells mediatedby OX40mAb24 using primary human NK cells from two separate donors asindicated. (16C-D) Lysis of Toledo B cells by NK cells from donors 464and 532 in the presence of rituximab, but not R347 human IgG1 isotypecontrol antibody. Technical replicates were conducted in duplicate.Error bars represent standard error of the mean.ADCC=antibody-drug-dependent-cytotoxicity; mAb=monoclonal antibody;NK=natural killer.

FIGS. 17A-B. Assessment of Natural Killer Cell-mediatedAntibody-Dependent Cellular Cytotoxicity of OX40mAb24, Experiment 5.Specific killing of OX40-expressing human CD4⁺ T cells mediated byOX40mAb24, using primary human NK cells from donors 601 (panel A) and602 (panel B) as indicated. Error bars represent standard error of themean. ADCC=antibody-drug-dependent-cytotoxicity; mAb=monoclonalantibody; NK=natural killer.

FIGS. 18A-B. Assessment of OX40mAb24 and 9B12 binding to purified humanC1q protein. The indicated concentrations of purified human C1q proteinwere injected onto the biosensor chip. Blank represents injections ofPBS/0.005% Tween 20 vehicle alone. Panel A: OX40mAb24 binding topurified human C1q. Panel B: 9B12 binding to purified human C1q.

FIGS. 19A-B. OX40mAb24 Activity in Cyno/Rhesus OX40-expressing JurkatNFκB-luciferase Clone B2 and LCL8664 Rhesus B-Cell Bioactivity Assays.Concentration-dependent induction of NFκB activity by OX40mAb24 (in RLU)in a cyno/rhesus OX40 expressing Jurkat NFκB-luciferase reporter cellline combined with rhesus B-cell line LCL8664. Data shown for 2independent assays with 4 replicates for each data point. Error barsrepresent standard error of the mean are not visible due to scale.RLU=relative light units.

FIGS. 20A-B. 9B12 Activity in Cyno/Rhesus OX40-expressing JurkatNFκB-luciferase Clone B2 and LCL8664 Rhesus B-Cell Bioactivity Assays.Concentration-dependent induction of NFκB activity by 9B12 (in RLU) in acyno/rhesus OX40 expressing Jurkat NFκB-luciferase reporter cell linecombined with rhesus B-cell line LCL8664. Data shown for 2 independentassays with 4 replicates for each data point. Error bars representstandard error of the mean. RLU=relative light units.

FIGS. 21A-B. OX40mAb24 and 9B12 Activity in Cyno/Rhesus OX40-expressingJurkat NFκB-luciferase Clone B2 and Fc Gamma Receptor-Expressing RhesusImmune Cells Bioactivity Assay. Concentration-dependent induction ofNFκB activity by OX40mAb24 (Panel A) and 9B12 (Panel B) (in RLU) in acyno/rhesus OX40 expressing Jurkat NFκB-luciferase reporter cell linecombined with Fcγ receptor-expressing rhesus immune cells. Tworeplicates per data point. RLU=relative light units.

FIGS. 22A-D. OX40mAb24 and 9B12 Cause Proliferation of Primary RhesusCD4 T Cells. OX40mAb24 (22A-B) and 9B12 (22C-D) induced cell division inprimary activated rhesus CD4⁺ T cells. Data is shown for 2 independentassays with triplicate wells. Error bars represent standard error of themean.

FIGS. 23A-B. Effect of OX40mAb24 and 9B12 on Growth of A375 Cells in aMouse Xenograft Model—Experiment 1. Six NOD/SCID mice in each group wereengrafted SC on Day 1 with A375 cells mixed with alloreactive human CD4⁺and CD8⁺ T cell lines at E:T ratio 1:6. OX40mAb24 (23A) and 9B12 (23B)and isotype control (23A-B) were administered IP on Days 3, 5, 7, 10 and12. Mean values of tumor volumes are shown. A comparison betweenOX40mAb24-treated (23A) or 9B12-treated (23B) and the isotypecontrol-treated animals was made on Day 25 and 18, respectively, andintergroup differences were analyzed for statistical significance by aMann-Whitney rank sum test. Error bars represent standard error of themean. *: TGI>68%, P<0.05 as compared to the isotype-control group.E:T=effector-to-target ratio; IP intraperitoneal; NOD/SCID=non-obesediabetic/severe combined immunodeficient; SC=subcutaneous; TGI=tumorgrowth inhibition.

FIGS. 24A-B. Effect of OX40mAb24 and 9B12 on Growth of A375 Cells in aMouse Xenograft Model—Experiment 2. Six NOD/SCID mice in each group wereengrafted SC on Day 1 with A375 cells mixed with alloreactive human CD4⁺and CD8⁺ T cell lines at E:T ratio 1:6. OX40mAb24 (24A) and 9B12 (24B)and isotype control (24A-B) were administered IP on Days 3, 5, 7, 10 and12. Mean values of tumor volumes are shown. A comparison betweenOX40mAb24-treated (12A) or 9B12-treated (12B) and the isotypecontrol-treated animals was made on Day 18, and intergroup differenceswere analyzed for statistical significance by a Mann-Whitney rank sumtest. Error bars represent standard error of the mean. #: TGI>75%,P<0.0004 as compared to the isotype-control group. *: TGI=53%, P<0.05 ascompared to the isotype-control group. E:T=effector-to-target ratio;IP=intraperitoneal; NOD/SCID=non-obese diabetic/severe combinedimmunodeficient; SC=subcutaneous; TGI=tumor growth inhibition.

FIG. 25. Effect of OX40mAb24 on Growth of A375 Cells in a MouseXenograft Model—Experiment 3. Six NOD/SCID mice in each group wereengrafted SC on Day 1 with A375 cells mixed with alloreactive human CD4⁺and CD8⁺ T cell lines at E:T ratio 1:6. OX40mAb24 was administered IP onDays 3, 6, 8, 10 and 13. Mean values of tumor volumes are shown. Acomparison between OX40mAb24-treated and the isotype control-treatedanimals was made on Day 28, and intergroup differences were analyzed forstatistical significance by a Mann-Whitney rank sum test. Error barsrepresent standard error of the mean. *: TGI=75%, P<0.05 as compared tothe isotype-control group; E:T=effector-to-target ratio;IP=intraperitoneal; NOD/SCID=non-obese diabetic/severe combinedimmunodeficient; SC=subcutaneous; TGI=tumor growth inhibition.

FIGS. 26A-B. Effect of OX86 mIgG2a on Growth of CT26 Cell Line in aMouse Syngeneic Model. Ten BALB/c mice in each group were inoculated SCon Day 1 with CT26 cells. Control (negative control) and test article(OX86 mIgG2a) were administered IP on Days 9 and 12 (arrows in (26A)).Mean (26A) and individual (26B) values of tumor volumes are shown. Acomparison between OX86 mIgG2a-treated and the negative control-treatedanimals was made, and intergroup differences were analyzed forstatistical significance by aone-way ANOVA using GraphPad Prism 6.0software. Error bars represent standard error of the mean. *: TGI>50%,P<0.0001 as compared to the isotype-control group on Day 21.IP=intraperitoneal; SC=subcutaneous; TGI=tumor growth inhibition.

FIGS. 27A-B. Effect of OX86 mIgG2a on Growth of CT26 Cell Line in aMouse Syngeneic Model. Twelve BALB/c mice in each group were inoculatedSC on Day 1 with CT26 cells. Test article (OX86 mIgG2a) was administeredIP on Days 13 and 16 (arrows in (27A)). Mean (27A) and individual (27B)values of tumor volumes are shown. A comparison between OX86mIgG2a-treated and the untreated control animals was made, andintergroup differences were analyzed for statistical significance by aone-way ANOVA using GraphPad Prism 6.0 software. Error bars representstandard error of the mean. *: TGI>50%, P<0.0001 as compared to theuntreated control group on Day 24. IP=intraperitoneal; SC=subcutaneous;TGI=tumor growth inhibition.

FIGS. 28A-B. Effect of OX86 mIgG2a on Growth of MCA205 Cell Line in aMouse Syngeneic Model. Fourteen C57BL/6 mice in each group wereinoculated SC on Day 1 with MCA205 cells. Control article (isotypecontrol) and test article (OX86 mIgG2a) were administered IP on Days 11and 14. Mean (28A) and individual (28B) values of tumor volumes areshown. A comparison between OX86 mIgG2a-treated and the isotypecontrol-treated animals was made, and intergroup differences wereanalyzed for statistical significance by a one-way ANOVA using GraphPadPrism 6.0 software. Error bars represent standard error of the mean. *:TGI>50%, P<0.0001 as compared to the isotype-control group on Day 27.IP=intraperitoneal; SC=subcutaneous; TGI=tumor growth inhibition.

FIGS. 29A-B. Effect of OX86 mIgG2a on Growth of 4T1 Cell Line in a MouseSyngeneic Model. Twelve BALB/c mice in each group were inoculated S onDay 1 with 4T1 cells. Control article (isotype control) and test article(OX86 mIgG2a) were administered IP on Days 13, 16, 20, and 23. Mean(29A) and individual (29B) values of tumor volumes are shown. Acomparison between OX86 mIgG2a-treated and the isotype control-treatedanimals was made, and intergroup differences were analyzed forstatistical significance by a one-way ANOVA using GraphPad Prism 6.0software. Error bars represent standard error of the mean.IP=intraperitoneal; SC=subcutaneous; TGI=tumor growth inhibition.

FIG. 30. Amino Acid Alignment of the Extracellular Domains of Human andMouse OX40 molecules. Human OX40 (NCBI reference sequence NP_003318.1)shares 60% sequence identity with mouse OX40 (NCBI reference sequenceNP_035789.1). The alignment was performed using the method of Clustal W.The extracellular domains of OX40 are detailed. Amino acids that differbetween human and mouse in the CRD3 domain are shown with arrows. Thetwo critical epitope residues L116 and A126 are boxed. CRD=cysteine-richdomain.

FIG. 31. Nomenclature and Schematic Representation of ChimericHuman/Mouse OX40 Variants. Chimeric human/mouse OX40 variants wereconstructed by swapping in or out various domains or residues of mouseOX40 (open) into human (solid) (KO) or of human OX40 amino acids intomouse OX40 (KI). Mutations for individual amino acids or combinationswere shown with a red (KO) and green (KI) arrows. CRD=cysteine-richdomain; KI=knock-in; KO=knock-out; TM=transmembrane domain.

FIGS. 32A-C. FACS Analysis of Binding of OX40mAb24 to ChimericHuman/Mouse OX40 Variants. All variants were transiently expressed using293F cells for binding characterization with FACS analysis usingOX40mAb24 and its parental mouse mAb, 9B12. Expression levels weremonitored using anti-human and mouse OX40 polyclonal antibodies. (A)Using domain-swapped chimeric variants, the CRD3 domain was identifiedas the epitope-containing domain. OX40mAb24 and 9B12 do not bind the KOvariants encoding for mouse CRD3 domain (KO_CRD3 and KO_CRD3+4), andrecognize the KI variant (KI_CRD3) encoding for human CRD3 domain. (B)Additionally, critical epitope residues were determined as L116 and A126in CRD3 domain by mutating individual or combinations of amino acids,which differ between human and mouse in the CRD3 domain (FIG. 1). Thebinding of OX40mAb24 and 9B12 was abolished when replacing humanresidues L116 and A126 with the mouse counterparts (KO_L116+A126). (C)The KI/gain-of-function variants confirm the importance of these twocritical residues. Grafting L116, A126, or the combination to mouse OX40led to the binding of OX40mAb24 and 9B12.

FIGS. 33A-D. Expression Levels of Ki67 and ICOS on Peripheral Blood andSplenic CD4⁺ T cells Following Administration of OX86 mIgG2a to NaïveMice. Seven naïve BALB/c mice in each group were inoculatedintraperitoneally on Day 1 with control articles (saline and NIP228IgG2a isotype control) and test article (OX86 mIgG2a) at the indicateddose levels. Blood (panels A and C) was collected on the indicated Daysand spleens from five groups were isolated on Day 10. Expression levelsof Ki67 (panel A and B) and ICOS (panels C and D) on CD4⁺ T cells weremeasured by flow cytometry. Mean values of the percentage of CD4 cellsin blood expressing Ki67 (panel A) and ICOS (panel C) are shown for eachgroup. Percentage of CD4 cells in the spleen expressing Ki67 (panel B)and ICOS (panel D) were plotted for each animal of individual groups.Error bars represent the standard error of the mean. *: P<0.05 aremarked in Panels A and C; P values are listed for each group withsignificance in Panels B and D.

FIGS. 34A-B. Correlation of Ki67 and ICOS Expression on Peripheral Bloodand Splenic CD4⁺ T cells Following Administration of OX86 mIgG2a toNaïve Mice. A comparison was made between the percentage of Ki67 andICOS positive CD4 T cells isolated from peripheral blood (panel A) andspleens (panel B) of individual animals shown in FIGS. 12A-D 10 daysfollowing treatment with control articles (saline and NIP228 IgG2aisotype control) and test article (OX86 mIgG2a) at the indicated doselevels. Measurements for individual mice were plotted. Linear regressionanalysis was performed using GraphPad Prism 6.0 software on theresulting group of data sets to determine a best-fit line for the data.The coefficient of determination (r2) and significance that the slope isnon-zero (P value) are provided for each graph.

FIGS. 35A-D. Expression Levels of Ki67 and ICOS on Peripheral Blood andSplenic CD8⁺ T cells Following Administration of OX86 mIgG2a to NaïveMice. Seven naïve BALB/c mice in each group were inoculatedintraperitoneally on Day 1 with control articles (saline and NIP228IgG2a isotype control) and test article (OX86 mIgG2a) at the indicateddose levels. Blood (panels A and C) was collected on the indicated Daysand spleens from five groups were isolated on Day 10. Expression levelsof Ki67 (panel A and B) and ICOS (panels C and D) on CD8⁺ T cells weremeasured by flow cytometry. Mean values of the percentage of CD8 cellsin blood expressing Ki67 (panel A) and ICOS (panel C) are shown for eachgroup. Percentage of CD8 cells in the spleen expressing Ki67 (panel B)and ICOS (panel D) were plotted for each animal of individual groups.Error bars represent the standard error of the mean. *: P<0.05 aremarked in Panels A and C; P values are listed for each group withsignificance in Panels B and D.

FIGS. 36A-B. Correlation of Ki67 and ICOS Expression on Peripheral Bloodand Splenic CD8⁺ T cells Following Administration of OX86 mIgG2a toNaïve Mice. A comparison was made between the percentage of Ki67 andICOS positive CD8⁺ T cells isolated from peripheral blood (panel A) andspleens (panel B) of individual animals shown in FIGS. 12A-D 10 daysfollowing treatment with control articles (saline and NIP228 IgG2aisotype control) and test article (OX86 mIgG2a) at the indicated doselevels. Measurements for individual mice were plotted. Linear regressionanalysis was performed using GraphPad Prism 6.0 software on theresulting group of data sets to determine a best-fit line for the data.The coefficient of determination (r2) and significance that the slope isnon-zero (P value) are provided for each graph.

FIGS. 37A-D. Effect of Mouse OX86 IgG2a on Growth of the CT26 Cell Linein Syngeneic Mouse Model Lacking Inhibitory (Fcgr2b−/−) or Activating(Fcer1g−/−) Fc Gamma Receptors. Groups of eight Balb/c mice geneticallyengineered to lack the inhibitory Fcγ receptor IIb (Fcgr2b−/−; panels Aand B) or the activating Fcγ receptors (Fcer1g−/−; panels C and D) wereinoculated SC on Day 1 with CT26 cells. Control articles(saline/untreated and OX86 mIgG1 D265A mutant/isotype control) and testarticle (OX86 mIgG2a) were administered IP on Days 4 and 7. Mean (panelA and C) and individual (panel B and D) tumor volumes are shown. Acomparison between OX86 mIgG2a-treated and the untreated and isotypecontrol-treated animals was made, and intergroup differences wereanalyzed for statistical significance by a one-way ANOVA using GraphPadPrism 6.0 software. Error bars represent standard error of the mean.SC=subcutaneous; IP=intraperitoneal; TGI=tumor growth inhibition

FIGS. 38A-D. Effect of Mouse OX86 IgG2a on Growth of the MCA205 CellLine in a Syngeneic Mouse Model Lacking Inhibitory (Fcgr2b−/−) orActivating (Fcer1g−/−) Fc Gamma Receptors. Groups of eight C57BL/6 micegenetically mutated to lack the inhibitory Fcγ receptor IIb (Fcgr2b−/−;panels A and B) or the activating Fcγ receptors (Fcer1g−/−; panels C andD were inoculated SC on Day 1 with MCA205 cells. Control articles(saline/untreated and OX86 mIgG1 D265A mutant/isotype control) and testarticle (mOX40L FP) were administered IP on Days 4 and 7. Mean (panel Aand C) and individual (panel B and D) tumor volumes are shown. Acomparison between mOX40L FP-treated and the untreated and isotypecontrol-treated animals was made, and intergroup differences wereanalyzed for statistical significance by a one-way ANOVA using GraphPadPrism 6.0 software. Error bars represent standard error of the mean. *:TGI>95%, P<0.023 as compared to the untreated and isotype-control groupson study day 20. SC=subcutaneous; IP=intraperitoneal; TGI=tumor growthinhibition.

FIGS. 39A-B. Expression Levels of Ki67 in CD4⁺ T cells Isolated fromPeripheral Blood, Spleen and CT26 Tumor Following Administration of OX86Mouse IgG2a to Mice Lacking Inhibitory (Fcgr2b−/−) or Activating(Fcer1g−/−) Fc Gamma Receptor. Groups of four Balb/c mice geneticallyengineered to lack the inhibitory Fcγ receptor IIb (Fcgr2b−/−; panels A)or the activating Fcγ receptors (Fcer1g−/−; panels B) were inoculated SCon Day 1 with CT26 cells; this study is independent of, but wasconducted similarly to, the study presented in FIGS. 12A-D. Controlarticles (saline/untreated and OX86 mIgG1 D265A mutant/isotype control)and test article (OX86 mIgG2a) were administered IP on Days 4 and 7.Blood, spleens and tumors were isolated on Day 14 for panel A and Day 13for panel B. Expression levels of Ki67 in CD4⁺ T cells were measured byflow cytometry. Symbols represent the percentage of Ki67 positive CD4⁺ Tcells from each tissue of individual mice; horizontal bar represents themean values for each group. Intergroup differences were analyzed forstatistical significance by a one-way ANOVA using GraphPad Prism 6.0software, and indicated with a horizontal bar with the calculated Pvalues. SC=subcutaneous; IP=intraperitoneal.

FIGS. 40A-B. Expression Levels of Ki67 in CD8⁺ T cells Isolated fromPeripheral Blood, Spleen and CT26 Tumor Following Administration of OX86Mouse IgG2a to Mice Lacking Inhibitory (Fcgr2b−/−) or Activating(Fcer1g−/−) Fc Gamma Receptors. Groups of four Balb/c mice, geneticallyengineered to lack the inhibitory Fcγ receptor IIb (Fcgr2b−/−; panels A)or the activating Fcγ receptors (Fcer1g−/−; panels B), were inoculatedSC on Day 1 with CT26 cells; this study is independent of but wasconducted similarly to the study presented in FIGS. 12A-D. Controlarticles (saline/untreated and OX86 mIgG1 D265A mutant/isotype control)and test article (OX86 mIgG2a) were administered IP on Days 4 and 7.Blood, spleens and tumor were isolated on Day 14 for panel A and Day 13for panel B. Expression levels of Ki67 on CD8⁺ T cells were measured byflow cytometry. Symbols represent the percentage of Ki67 positive CD8⁺ Tcells from each tissue of individual mice; horizontal bar represents themean values. Intergroup differences were analyzed for statisticalsignificance by a one-way ANOVA using GraphPad Prism 6.0 software, andindicated with a horizontal bar with the calculated P values.SC=subcutaneous; IP=intraperitoneal.

FIGS. 41A-B. Expression Levels of Ki67 in CD4 T cells Isolated fromDraining Lymph Node, Spleen and MCA205 Tumor Following Administration ofOX86 Mouse IgG2a to Mice Lacking Inhibitory (Fcgr2b^(−/−)) or Activating(Fcer1g^(−/−)) Fc Gamma Receptors. Groups of four C57BL/6 mice,genetically engineered to lack the inhibitory Fcγ receptor IIb(Fcgr2b^(−/−); panels A) or the activating Fcγ receptors (Fcer1g^(−/−);panels B), were inoculated SC on Day 1 with MCA205 cells; study isindependent but conducted similarly to the study presented in FIGS.12A-D. Control articles (saline/untreated and OX86 mIgG1 D265Amutant/isotype control) and test article (OX86 mIgG2a) were administeredIP on Days 4 and 7. Draining lymph nodes, spleens and tumor wereisolated on Day 20 for panel B. Expression levels of Ki67 in CD4⁺ Tcells were measured by flow cytometry. Symbols represent the percentageof Ki67 positive CD4⁺ T cells from each tissue of individual mice;horizontal bar represents the mean values. Intergroup differences wereanalyzed for statistical significance by a one-way ANOVA using GraphPadPrism 6.0 software, and indicated with a horizontal bar with thecalculated P values. SC=subcutaneous; IP=intraperitoneal.

FIGS. 42A-B. Expression Levels of Ki67 in CD8 T cells Isolated fromDraining Lymph Node, Spleen and MCA205 Tumor Following Administration ofOX86 Mouse IgG2a to Mice Lacking Inhibitory (Fcgr2b−/−) or Activating(Fcer1g−/−) Fc Gamma Receptors. Groups of four C57BL/6 mice, geneticallyengineered to lack the inhibitory Fcγ receptor IIb (Fcgr2b−/−; panels A)or the activating Fcγ receptors (Fcer1g−/−; panels B), were inoculatedSC on Day 1 with MCA205 cells; this study is independent of but wasconducted similarly to the study presented in FIGS. 12A-D. Controlarticles (saline and mOX40L FP D265A mutant control) and test article(OX86 mIgG2a) were administered IP on Days 4 and 7. Draining lymphnodes, spleens and tumor were isolated on Day 20. Expression levels ofKi67 on CD8⁺ T cells were measured by flow cytometry. Symbols representthe percentage of Ki67 positive CD8⁺ T cells from each tissue ofindividual mice; horizontal bar represents the mean values. Intergroupdifferences were analyzed for statistical significance by a one-wayANOVA using GraphPad Prism 6.0 software, and indicated with a horizontalbar with the calculated P values. SC=subcutaneous; IP=intraperitoneal

FIGS. 43A-B. Reduction of Regulatory T Cells by OX40mAb24. Panel A:Percentages of human CD4⁺ Foxp3⁺ Treg cells measured in the peripheralblood of mice engrafted with human immune cells just prior to KLHimmunization and treatment with NIP228 huIgG1 control (huIgG1) orOX40mAb24. No mAb indicates no immunization and no mAb treatment. PanelB: Percentages of human CD4⁺FoxP3⁺ Treg cells in the peripheral blood ofmice in the same groups after immunization and treatment with mAbs.Statistical p values are from one-way ANOVA. Hu=human; mAb=monoclonalantibody; Treg=regulatory T cells

FIGS. 44A-D. Expansion of Effector and Memory CD4 T Cells Relative toRegulatory T Cells after OX40mAb24 Treatment. Ratios of human CD4⁺ Tcells to human Treg cells in the peripheral blood of mice engrafted withhuman immune cells either pre-treatment (left) or post-treatment (right)with KLH immunization followed by NIP228 huIgG1 mAb (huIgG1) orOX40mAb24, as indicated, for Total CD4⁺ (Panel A); CD4⁺ effector (Teff)(Panel B); CD4⁺ effector memory (Tem) (Panel C); and CD4⁺ central memoryT cells (Tcm) (Panel D). No mAb indicates no immunization as well as nomAb treatment. Statistical p values are from one-way ANOVA. Hu=human;mAb=monoclonal antibody; Tcm=central memory Tcell; Teff=effector T cell;Tem=effector memory T cell

FIGS. 45A-B. Increased CD8⁺ Effector to Regulatory T Cell Ratio in MiceTreated with OX40mAb24. Ratios of human CD8⁺ effector T cells to humanTreg cells in the peripheral blood of mice engrafted with human immunecells either pre-treatment (left) or post-treatment (right) with KLHimmunization followed by NIP228 huIgG1 mAb (huIgG1) or OX40mAb24, asindicated. No mAb indicates no immunization as well as no mAb treatment.Statistical p value from one-way ANOVA comparing all groups isindicated. Hu=human; mAb=monoclonal antibody; Teff=effector T cell;Treg=regulatory T cell

FIGS. 46A-C. Increased CD25 (IL-2 Receptor) Levels on CD8⁺ T Cells inMice Treated with OX40mAb24. Percentage of human CD25 (IL-2 receptor)positive cells among human CD8⁺ T cells in the peripheral blood of miceengrafted with human immune cells either post-treatment (left) orpre-treatment (right) with no treatment (no mAb), or KLH immunizationfollowed by NIP228 huIgG1 mAb (huIgG1) or OX40MAB24, as indicated, for(A) Total CD8⁺, (B) CD8⁺ effector (Teff), (C) CD8⁺ effector memory (Tem)T cells. No mAb indicates no immunization as well as no mAb treatment.Statistical p values are from one-way ANOVA. Hu=human; mAb=monoclonalantibody; Teff=effector T cell; Tem=effector memory T cell.

DETAILED DESCRIPTION

Engagement of the OX40 receptor on T cells, e.g., CD4⁺ T cells during,or shortly after, priming by an antigen results in an increased responseof the T cells, e.g., CD4⁺ T cells to the antigen. In the context of thepresent disclosure, the term “engagement” refers to binding to andstimulation of at least one activity mediated by the OX40 receptor. Forexample, engagement of the OX40 receptor on antigen specific T cells,e.g., CD4⁺ T cells can result in increased T cell proliferation ascompared to the response to antigen alone, and increased cytokineproduction. The elevated response to the antigen can be maintained for aperiod of time substantially longer than in the absence of OX40 receptorengagement. Thus, stimulation via the OX40 receptor enhances the antigenspecific immune response by boosting T cell recognition of antigens,e.g., tumor antigens.

OX40 agonists can enhance antigen specific immune responses in asubject, such as a human subject, when administered to the subjectduring or shortly after priming of T cells by an antigen. OX40 agonistsinclude OX40 ligand (“OX40L”), such as soluble OX40L fusion proteins andanti-OX40 antibodies or fragments thereof. A specific example is ahumanized antibody that specifically binds to OX40, thereby triggeringsignaling. A collection of humanized anti-OX40 monoclonal antibodies areprovided by this disclosure. Also described are nucleic acids includingpolynucleotide sequences that encode such antibodies. This disclosurealso provides methods for enhancing an antigen specific immune responsein a subject using humanized anti-OX40 monoclonal antibodies.

Definitions

It is to be noted that the term “a” or “an” entity refers to one or moreof that entity; for example, “a binding molecule,” is understood torepresent one or more binding molecules. As such, the terms “a” (or“an”), “one or more,” and “at least one” can be used interchangeablyherein.

Furthermore, “and/or” where used herein is to be taken as specificdisclosure of each of the two specified features or components with orwithout the other. Thus, the term and/or” as used in a phrase such as “Aand/or B” herein is intended to include “A and B,” “A or B,” “A”(alone), and “B” (alone) Likewise, the term “and/or” as used in a phrasesuch as “A, B, and/or C” is intended to encompass each of the followingembodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; Aand B; B and C; A (alone); B (alone); and C (alone).

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure is related. For example, the ConciseDictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed.,2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed.,1999, Academic Press; and the Oxford Dictionary Of Biochemistry AndMolecular Biology, Revised, 2000, Oxford University Press, provide oneof skill with a general dictionary of many of the terms used in thisdisclosure.

Units, prefixes, and symbols are denoted in their Systeme Internationalde Unites (SI) accepted form. Numeric ranges are inclusive of thenumbers defining the range. Unless otherwise indicated, amino acidsequences are written left to right in amino to carboxy orientation. Theheadings provided herein are not limitations of the various aspects oraspects of the disclosure, which can be had by reference to thespecification as a whole. Accordingly, the terms defined immediatelybelow are more fully defined by reference to the specification in itsentirety.

As used herein, the term “non-naturally occurring” substance,composition, entity, and/or any combination of substances, compositions,or entities, or any grammatical variants thereof, is a conditional termthat explicitly excludes, but only excludes, those forms of thesubstance, composition, entity, and/or any combination of substances,compositions, or entities that are, or that might be at any time,determined or interpreted by a judge or an administrative or judicialbody to be, “naturally-occurring.”

As used herein, the term “polypeptide” is intended to encompass asingular “polypeptide” as well as plural “polypeptides,” and refers to amolecule composed of monomers (amino acids) linearly linked by amidebonds (also known as peptide bonds). The term “polypeptide” refers toany chain or chains of two or more amino acids, and does not refer to aspecific length of the product. Thus, peptides, dipeptides, tripeptides,oligopeptides, “protein,” “amino acid chain,” or any other term used torefer to a chain or chains of two or more amino acids are includedwithin the definition of “polypeptide,” and the term “polypeptide” canbe used instead of, or interchangeably with any of these terms. The term“polypeptide” is also intended to refer to the products ofpost-expression modifications of the polypeptide, including withoutlimitation glycosylation, acetylation, phosphorylation, amidation, andderivatization by known protecting/blocking groups, proteolyticcleavage, or modification by non-naturally occurring amino acids. Apolypeptide can be derived from a biological source or produced byrecombinant technology, but is not necessarily translated from adesignated nucleic acid sequence. It can be generated in any manner,including by chemical synthesis.

A polypeptide as disclosed herein can be of a size of about 3 or more, 5or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100or more, 200 or more, 500 or more, 1,000 or more, or 2,000 or more aminoacids. Polypeptides can have a defined three-dimensional structure,although they do not necessarily have such structure. Polypeptides witha defined three-dimensional structure are referred to as folded, andpolypeptides which do not possess a defined three-dimensional structure,but rather can adopt a large number of different conformations, and arereferred to as unfolded. As used herein, the term glycoprotein refers toa protein coupled to at least one carbohydrate moiety that is attachedto the protein via an oxygen-containing or a nitrogen-containing sidechain of an amino acid, e.g., a serine or an asparagine.

By an “isolated” polypeptide or a fragment, variant, or derivativethereof is intended a polypeptide that is not in its natural milieu. Noparticular level of purification is required. For example, an isolatedpolypeptide can be removed from its native or natural environment.Recombinantly produced polypeptides and proteins expressed in host cellsare considered isolated as disclosed herein, as are native orrecombinant polypeptides which have been separated, fractionated, orpartially or substantially purified by any suitable technique.

As used herein, the term “non-naturally occurring” polypeptide, or anygrammatical variants thereof, is a conditional term that explicitlyexcludes, but only excludes, those forms of the polypeptide that are, orthat might be at any time, determined or interpreted by a judge or anadministrative or judicial body to be, “naturally-occurring.”

Other polypeptides disclosed herein are fragments, derivatives, analogs,or variants of the foregoing polypeptides, and any combination thereof.The terms “fragment,” “variant,” “derivative” and “analog” as disclosedherein include any polypeptides which retain at least some of theproperties of the corresponding native antibody or polypeptide, forexample, specifically binding to an antigen. Fragments of polypeptidesinclude, for example, proteolytic fragments, as well as deletionfragments, in addition to specific antibody fragments discussedelsewhere herein. Variants of, e.g., a polypeptide include fragments asdescribed above, and also polypeptides with altered amino acid sequencesdue to amino acid substitutions, deletions, or insertions. In certainaspects, variants can be non-naturally occurring. Non-naturallyoccurring variants can be produced using art-known mutagenesistechniques. Variant polypeptides can comprise conservative ornon-conservative amino acid substitutions, deletions or additions.Derivatives are polypeptides that have been altered so as to exhibitadditional features not found on the original polypeptide. Examplesinclude fusion proteins. Variant polypeptides can also be referred toherein as “polypeptide analogs.” As used herein a “derivative” of apolypeptide can also refer to a subject polypeptide having one or moreamino acids chemically derivatized by reaction of a functional sidegroup. Also included as “derivatives” are those peptides that containone or more derivatives of the twenty standard amino acids. For example,4-hydroxyproline can be substituted for proline; 5-hydroxylysine can besubstituted for lysine; 3-methylhistidine can be substituted forhistidine; homoserine can be substituted for serine; and ornithine canbe substituted for lysine.

A “conservative amino acid substitution” is one in which one amino acidis replaced with another amino acid having a similar side chain.Families of amino acids having similar side chains have been defined inthe art, including basic side chains (e.g., lysine, arginine,histidine), acidic side chains (e.g., aspartic acid, glutamic acid),uncharged polar side chains (e.g., asparagine, glutamine, serine,threonine, tyrosine, cysteine), nonpolar side chains (e.g., glycine,alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). For example, substitution of aphenylalanine for a tyrosine is a conservative substitution. In certainembodiments, conservative substitutions in the sequences of thepolypeptides and antibodies of the present disclosure do not abrogatethe binding of the polypeptide or antibody containing the amino acidsequence, to the antigen to which the binding molecule binds. Methods ofidentifying nucleotide and amino acid conservative substitutions whichdo not eliminate antigen-binding are well-known in the art (see, e.g.,Brummell et al., Biochem. 32: 1180-1 187 (1993); Kobayashi et al.,Protein Eng. 12(10):879-884 (1999); and Burks et al., Proc. Natl. Acad.Sci. USA 94:412-417 (1997)).

The term “polynucleotide” is intended to encompass a singular nucleicacid as well as plural nucleic acids, and refers to an isolated nucleicacid molecule or construct, e.g., messenger RNA (mRNA), cDNA, or plasmidDNA (pDNA). A polynucleotide can comprise a conventional phosphodiesterbond or a non-conventional bond (e.g., an amide bond, such as found inpeptide nucleic acids (PNA)). The terms “nucleic acid” or “nucleic acidsequence” refer to any one or more nucleic acid segments, e.g., DNA orRNA fragments, present in a polynucleotide.

By an “isolated” nucleic acid or polynucleotide is intended any form ofthe nucleic acid or polynucleotide that is separated from its nativeenvironment. For example, gel-purified polynucleotide, or a recombinantpolynucleotide encoding a polypeptide contained in a vector would beconsidered to be “isolated.” Also, a polynucleotide segment, e.g., a PCRproduct, which has been engineered to have restriction sites for cloningis considered to be “isolated.” Further examples of an isolatedpolynucleotide include recombinant polynucleotides maintained inheterologous host cells or purified (partially or substantially)polynucleotides in a non-native solution such as a buffer or saline.Isolated RNA molecules include in vivo or in vitro RNA transcripts ofpolynucleotides, where the transcript is not one that would be found innature.

Isolated polynucleotides or nucleic acids further include such moleculesproduced synthetically. In addition, polynucleotide or a nucleic acidcan be or can include a regulatory element such as a promoter, ribosomebinding site, or a transcription terminator.

As used herein, a “non-naturally occurring” polynucleotide, or anygrammatical variants thereof, is a conditional definition thatexplicitly excludes, but only excludes, those forms of thepolynucleotide that are, or that might be at any time, determined orinterpreted by a judge or an administrative or judicial body to be,“naturally-occurring.”

As used herein, a “coding region” is a portion of nucleic acid whichconsists of codons translated into amino acids. Although a “stop codon”(TAG, TGA, or TAA) is not translated into an amino acid, it can beconsidered to be part of a coding region, but any flanking sequences,for example promoters, ribosome binding sites, transcriptionalterminators, introns, and the like, are not part of a coding region. Twoor more coding regions can be present in a single polynucleotideconstruct, e.g., on a single vector, or in separate polynucleotideconstructs, e.g., on separate (different) vectors. Furthermore, anyvector can contain a single coding region, or can comprise two or morecoding regions, e.g., a single vector can separately encode animmunoglobulin heavy chain variable region and an immunoglobulin lightchain variable region. In addition, a vector, polynucleotide, or nucleicacid can include heterologous coding regions, either fused or unfused toanother coding region. Heterologous coding regions include withoutlimitation, those encoding specialized elements or motifs, such as asecretory signal peptide or a heterologous functional domain.

In certain embodiments, the polynucleotide or nucleic acid is DNA. Inthe case of DNA, a polynucleotide comprising a nucleic acid whichencodes a polypeptide normally can include a promoter and/or othertranscription or translation control elements operably associated withone or more coding regions. An operable association is when a codingregion for a gene product, e.g., a polypeptide, is associated with oneor more regulatory sequences in such a way as to place expression of thegene product under the influence or control of the regulatorysequence(s). Two DNA fragments (such as a polypeptide coding region anda promoter associated therewith) are “operably associated” if inductionof promoter function results in the transcription of mRNA encoding thedesired gene product and if the nature of the linkage between the twoDNA fragments does not interfere with the ability of the expressionregulatory sequences to direct the expression of the gene product orinterfere with the ability of the DNA template to be transcribed. Thus,a promoter region would be operably associated with a nucleic acidencoding a polypeptide if the promoter was capable of effectingtranscription of that nucleic acid. The promoter can be a cell-specificpromoter that directs substantial transcription of the DNA inpredetermined cells. Other transcription control elements, besides apromoter, for example enhancers, operators, repressors, andtranscription termination signals, can be operably associated with thepolynucleotide to direct cell-specific transcription.

A variety of transcription control regions are known to those skilled inthe art. These include, without limitation, transcription controlregions which function in vertebrate cells, such as, but not limited to,promoter and enhancer segments from cytomegaloviruses (the immediateearly promoter, in conjunction with intron-A), simian virus 40 (theearly promoter), and retroviruses (such as Rous sarcoma virus). Othertranscription control regions include those derived from vertebrategenes such as actin, heat shock protein, bovine growth hormone andrabbit B-globin, as well as other sequences capable of controlling geneexpression in eukaryotic cells. Additional suitable transcriptioncontrol regions include tissue-specific promoters and enhancers as wellas lymphokine-inducible promoters (e.g., promoters inducible byinterferons or interleukins).

Similarly, a variety of translation control elements are known to thoseof ordinary skill in the art. These include, but are not limited toribosome binding sites, translation initiation and termination codons,and elements derived from picornaviruses (particularly an internalribosome entry site, or IRES, also referred to as a CITE sequence).

In other embodiments, a polynucleotide can be RNA, for example, in theform of messenger RNA (mRNA), transfer RNA, or ribosomal RNA.

Polynucleotide and nucleic acid coding regions can be associated withadditional coding regions which encode secretory or signal peptides,which direct the secretion of a polypeptide encoded by a polynucleotideas disclosed herein. According to the signal hypothesis, proteinssecreted by mammalian cells have a signal peptide or secretory leadersequence which is cleaved from the mature protein once export of thegrowing protein chain across the rough endoplasmic reticulum has beeninitiated. Those of ordinary skill in the art are aware thatpolypeptides secreted by vertebrate cells can have a signal peptidefused to the N-terminus of the polypeptide, which is cleaved from thecomplete or “full length” polypeptide to produce a secreted or “mature”form of the polypeptide. In certain embodiments, the native signalpeptide, e.g., an immunoglobulin heavy chain or light chain signalpeptide is used, or a functional derivative of that sequence thatretains the ability to direct the secretion of the polypeptide that isoperably associated with it. Alternatively, a heterologous mammaliansignal peptide, or a functional derivative thereof, can be used. Forexample, the wild-type leader sequence can be substituted with theleader sequence of human tissue plasminogen activator (TPA) or mouseβ-glucuronidase.

Disclosed herein are certain binding molecules, or antigen-bindingfragments, variants, or derivatives thereof. Unless specificallyreferring to full-sized antibodies, the term “binding molecule”encompasses full-sized antibodies as well as antigen-binding subunits,fragments, variants, analogs, or derivatives of such antibodies, e.g.,engineered antibody molecules or fragments that bind antigen in a mannersimilar to antibody molecules, but which use a different scaffold.

As used herein, the term “binding molecule” refers in its broadest senseto a molecule that specifically binds to a receptor, e.g., an epitope oran antigenic determinant. As described further herein, a bindingmolecule can comprise one of more “antigen binding domains” describedherein. A non-limiting example of a binding molecule is an antibody orfragment thereof that retains antigen-specific binding.

As used herein, the terms “binding domain,” “receptor binding domain,”or “antigen binding domain” refer to a region of a binding molecule thatis necessary and sufficient to specifically bind to an epitope. Forexample, an “Fv,” e.g., a variable heavy chain and variable light chainof an antibody, either as two separate polypeptide subunits or as asingle chain, is considered to be a “binding domain.” Other bindingdomains include, without limitation, the variable heavy chain (VHH) ofan antibody derived from a camelid species, or six immunoglobulincomplementarity determining regions (CDRs) expressed in a heterologousframework, e.g., a different germline or species, or in a differentscaffold, e.g., a fibronectin scaffold. A “binding molecule” asdescribed herein can include one, two, three, four, five, six, seven,eight, nine, ten, eleven, twelve or more “antigen binding domains.”

The terms “antibody” and “immunoglobulin” can be used interchangeablyherein. An antibody (or a fragment, variant, or derivative thereof asdisclosed herein) includes at least the variable domain of a heavy chain(for camelid species) or at least the variable domains of a heavy chainand a light chain. Basic immunoglobulin structures in vertebrate systemsare relatively well understood. See, e.g., Harlow et al., Antibodies: ALaboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988).Unless otherwise stated, the term “antibody” encompasses anythingranging from a small antigen-binding fragment of an antibody to a fullsized antibody, e.g., an IgG antibody that includes two complete heavychains and two complete light chains, an IgA antibody that includes fourcomplete heavy chains and four complete light chains and optionallyincludes a J chain and/or a secretory component, or an IgM antibody thatincludes ten or twelve complete heavy chains and ten or twelve completelight chains and optionally includes a J chain.

As will be discussed in more detail below, the term “immunoglobulin”comprises various broad classes of polypeptides that can bedistinguished biochemically. Those skilled in the art will appreciatethat heavy chains are classified as gamma, mu, alpha, delta, or epsilon,(γ, μ, α, δ, ε) with some subclasses among them (e.g., γ1-γ4 or α1-α2)).It is the nature of this chain that determines the “class” of theantibody as IgG, IgM, IgA IgG, or IgE, respectively. The immunoglobulinsubclasses (isotypes) e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, IgA₂, etc. arewell characterized and are known to confer functional specialization.Modified versions of each of these classes and isotypes are readilydiscernible to the skilled artisan in view of the instant disclosureand, accordingly, are within the scope of this disclosure.

Light chains are classified as either kappa or lambda (κ, λ). Each heavychain class can be associated with either a kappa or lambda light chain.In general, the light and heavy chains are covalently bonded to eachother, and the “tail” portions of the two heavy chains are bonded toeach other by covalent disulfide linkages or non-covalent linkages whenthe immunoglobulins are generated either by hybridomas, B cells orgenetically engineered host cells. In the heavy chain, the amino acidsequences run from an N-terminus at the forked ends of the Yconfiguration to the C-terminus at the bottom of each chain. The basicstructure of certain antibodies, e.g., IgG antibodies, includes twoheavy chain subunits and two light chain subunits covalently connectedvia disulfide bonds to form a “Y” structure, also referred to herein asan “H2L2” structure.

Both the light and heavy chains are divided into regions of structuraland functional homology. The terms “constant” and “variable” are usedfunctionally. In this regard, it will be appreciated that the variabledomains of both the variable light (VL) and variable heavy (VH) chainportions determine antigen recognition and specificity. Conversely, theconstant domains of the light chain (CL) and the heavy chain (CH1, CH2or CH3) confer biological properties such as secretion, transplacentalmobility, Fc receptor binding, complement binding, and the like. Byconvention the numbering of the constant region domains increases asthey become more distal from the antigen binding site or amino-terminusof the antibody. The N-terminal portion is a variable region and at theC-terminal portion is a constant region; the CH3 (or CH4 in the case ofIgM) and CL domains actually comprise the carboxy-terminus of the heavyand light chain, respectively.

As indicated above, a binding domain, e.g., an antibody variable regionallows the binding molecule to selectively recognize and specificallybind a receptor, or an epitope on an antigen. That is, the VL domain andVH domain, or subset of the complementarity determining regions (CDRs),of a binding molecule, e.g., an antibody combine to form the variableregion that defines a three dimensional antigen binding site. Morespecifically, the antigen binding site is defined by three CDRs on eachof the VH and VL chains. Certain antibodies form larger structures. Forexample, IgA can form a molecule that includes two H2L2 units, a Jchain, and a secretory component, all covalently connected via disulfidebonds, and IgM can form a pentameric or hexameric molecule that includesfive or six H2L2 units and optionally a J chain covalently connected viadisulfide bonds.

The six “complementarity determining regions” or “CDRs” present in anantibody antigen-binding domain are short, non-contiguous sequences ofamino acids that are specifically positioned to form the binding domainas the antibody assumes its three dimensional configuration in anaqueous environment. The remainder of the amino acids in the bindingdomain, referred to as “framework” or “FW” regions, show lessinter-molecular variability. The framework regions largely adopt aβ-sheet conformation and the CDRs form loops which connect, and in somecases form part of, the β-sheet structure. Thus, framework regions actto form a scaffold that provides for positioning the CDRs in correctorientation by inter-chain, non-covalent interactions. The bindingdomain formed by the positioned CDRs defines a surface complementary tothe epitope on the immunoreactive antigen. This complementary surfacepromotes the non-covalent binding of the antibody to its cognateepitope. The amino acids that make up the CDRs and the frameworkregions, respectively, can be readily identified for any given heavy orlight chain variable region by one of ordinary skill in the art, sincethey have been defined in various different ways (see, “Sequences ofProteins of Immunological Interest,” Kabat, E., et al., U.S. Departmentof Health and Human Services, (1983); and Chothia and Lesk, J. Mol.Biol., 196:901-917 (1987), which are incorporated herein by reference intheir entireties).

In the case where there are two or more definitions of a term which isused and/or accepted within the art, the definition of the term as usedherein is intended to include all such meanings unless explicitly statedto the contrary. A specific example is the use of the term“complementarity determining region” (“CDR”) to describe thenon-contiguous antigen combining sites found within the variable regionof both heavy and light chain polypeptides. These particular regionshave been described, for example, by Kabat et al., U.S. Dept. of Healthand Human Services, “Sequences of Proteins of Immunological Interest”(1983) and by Chothia et al., J. Mol. Biol. 196:901-917 (1987), whichare incorporated herein by reference. The Kabat and Chothia definitionsinclude overlapping or subsets of amino acids when compared against eachother. Nevertheless, application of either definition (or otherdefinitions known to those of ordinary skill in the art) to refer to aCDR of an antibody or variant thereof is intended to be within the scopeof the term as defined and used herein, unless otherwise indicated. Theappropriate amino acids which encompass the CDRs as defined by each ofthe above cited references are set forth below in Table 1 as acomparison. The exact amino acid numbers which encompass a particularCDR will vary depending on the sequence and size of the CDR. Thoseskilled in the art can routinely determine which amino acids comprise aparticular CDR given the variable region amino acid sequence of theantibody.

TABLE 1 CDR Definitions¹ Kabat Chothia VH CDR1 31-35 26-32 VH CDR2 50-6552-58 VH CDR3  95-102  95-102 VL CDR1 24-34 26-32 VL CDR2 50-56 50-52 VLCDR3 89-97 91-96 ¹Numbering of all CDR definitions in Table 1 isaccording to the numbering conventions set forth by Kabat et al. (seebelow).

Kabat et al. also defined a numbering system for antibody heavy andlight chains, e.g., antibody variable domain sequences that isapplicable to any antibody. One of ordinary skill in the art canunambiguously assign this system of “Kabat numbering” to any variabledomain sequence, without reliance on any experimental data beyond thesequence itself. As used herein, “Kabat numbering” refers to thenumbering system set forth by Kabat et al., U.S. Dept. of Health andHuman Services, “Sequence of Proteins of Immunological Interest” (1983).Unless use of the Kabat numbering system is explicitly noted, however,consecutive numbering is used for all amino acid sequences in thisdisclosure.

Binding molecules, e.g., antibodies or antigen-binding fragments,variants, or derivatives thereof include, but are not limited to,polyclonal, monoclonal, human, humanized, or chimeric antibodies, singlechain antibodies, epitope-binding fragments, e.g., Fab, Fab′ andF(ab′)₂, Fd, Fvs, single-chain Fvs (scFv), single-chain antibodies,disulfide-linked Fvs (sdFv), fragments comprising either a VL or VHdomain, fragments produced by a Fab expression library. ScFv moleculesare known in the art and are described, e.g., in U.S. Pat. No.5,892,019. Immunoglobulin or antibody molecules encompassed by thisdisclosure can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY),class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass ofimmunoglobulin molecule.

By “specifically binds,” it is generally meant that a binding molecule,e.g., an antibody or fragment, variant, or derivative thereof binds toan epitope via its antigen binding domain, and that the binding entailssome complementarity between the antigen binding domain and the epitope.According to this definition, a binding molecule is said to“specifically bind” to an epitope when it binds to that epitope via itsantigen binding domain more readily than it would bind to a random,unrelated epitope. The term “specificity” is used herein to qualify therelative affinity by which a certain binding molecule binds to a certainepitope. For example, binding molecule “A” can be deemed to have ahigher specificity for a given epitope than binding molecule “B,” orbinding molecule “A” can be said to bind to epitope “C” with a higherspecificity than it has for related epitope “D.”

A binding molecule, e.g., an antibody or fragment, variant, orderivative thereof disclosed herein can be said to bind a target antigenwith an off rate (k(off)) of less than or equal to, e.g., 5×10⁻² sec⁻¹,10⁻² sec⁻¹, 5×10⁻³ sec⁻¹, 10⁻³ sec⁻¹, 5×10⁻⁴ sec⁻¹, 10⁻⁴ sec⁻¹, 5×10⁻⁵sec⁻¹, or 10⁻⁵ sec⁻¹5×10⁻⁶ sec⁻¹, 10⁻⁶ sec⁻¹, 5×10⁻⁷ sec⁻¹ or 10⁻⁷sec⁻¹.

A binding molecule, e.g., an antibody or antigen-binding fragment,variant, or derivative disclosed herein can be said to bind a targetantigen with an on rate (k(on)) of greater than or equal to, e.g., 10³M⁻¹ sec⁻¹, 5×10³M⁻¹ sec⁻¹, 10⁴ M⁻¹ sec⁻¹, 5×10⁴ M⁻¹ sec⁻¹, 10⁵ M⁻¹sec⁻¹, 5×10⁵ M⁻¹ sec⁻¹, 10⁶ M⁻¹ sec⁻¹, or 5×10⁶ M⁻¹ sec⁻¹ or 10⁷ M⁻¹sec⁻¹.

A binding molecule, e.g., an antibody or fragment, variant, orderivative thereof is said to competitively inhibit binding of areference antibody or antigen binding fragment to a given epitope if itpreferentially binds to that epitope to the extent that it blocks, tosome degree, binding of the reference antibody or antigen bindingfragment to the epitope. Competitive inhibition can be determined by anymethod known in the art, for example, competition ELISA assays. Abinding molecule can be said to competitively inhibit binding of thereference antibody or antigen binding fragment to a given epitope by atleast 90%, at least 80%, at least 70%, at least 60%, or at least 50%.

As used herein, the term “affinity” refers to a measure of the strengthof the binding of an individual epitope with one or more bindingdomains, e.g., of an immunoglobulin molecule. See, e.g., Harlow et al.,Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press,2nd ed. 1988) at pages 27-28. As used herein, the term “avidity” refersto the overall stability of the complex between a population of bindingdomains and an antigen. See, e.g., Harlow at pages 29-34. Avidity isrelated to both the affinity of individual binding domains in thepopulation with specific epitopes, and also the valencies of theimmunoglobulins and the antigen. For example, the interaction between abivalent monoclonal antibody and an antigen with a highly repeatingepitope structure, such as a polymer, would be one of high avidity. Aninteraction between a between a bivalent monoclonal antibody with areceptor present at a high density on a cell surface would also be ofhigh avidity.

Binding molecules or antigen-binding fragments, variants or derivativesthereof as disclosed herein can also be described or specified in termsof their cross-reactivity. As used herein, the term “cross-reactivity”refers to the ability of a binding molecule, e.g., an antibody orfragment, variant, or derivative thereof, specific for one antigen, toreact with a second antigen; a measure of relatedness between twodifferent antigenic substances. Thus, a binding molecule is crossreactive if it binds to an epitope other than the one that induced itsformation. The cross reactive epitope generally contains many of thesame complementary structural features as the inducing epitope, and insome cases, can actually fit better than the original.

A binding molecule, e.g., an antibody or fragment, variant, orderivative thereof can also be described or specified in terms of theirbinding affinity to an antigen. For example, a binding molecule can bindto an antigen with a dissociation constant or K_(D) no greater than,e.g., 5×10⁻² M, 10⁻²M, 5×10⁻³M, 10⁻³M, 5×10⁻⁴M, 10⁻⁴M, 5×10⁻⁵M, 10⁻⁵M,5×10⁻⁶M, 10⁻⁶M, 5×10⁻⁷ M, 10⁻⁷M, 5×10⁻⁸M, 10⁻⁸M, 5×10⁻⁹M, 10⁻⁹M,5×10⁻¹⁰M, 10⁻¹⁰M, 5×10⁻¹¹M, 10⁻¹¹M, 5×10⁻¹²M, 10⁻¹² M, 5×10⁻¹³M, 10⁻¹³M,5×10⁻¹⁴M, 10⁻¹⁴M, 5×10⁻¹⁵M, or 10⁻¹⁵M.

Antibody fragments including single-chain antibodies or other bindingdomains can exist alone or in combination with one or more of thefollowing: hinge region, CH1, CH2, CH3, or CH4 domains, J chain, orsecretory component. Also included are antigen-binding fragments thatcan include any combination of variable region(s) with one or more of ahinge region, CH1, CH2, CH3, or CH4 domains, a J chain, or a secretorycomponent. Binding molecules, e.g., antibodies, or antigen-bindingfragments thereof can be from any animal origin including birds andmammals. The antibodies can be human, murine, donkey, rabbit, goat,guinea pig, camel, llama, horse, or chicken antibodies. In anotherembodiment, the variable region can be condricthoid in origin (e.g.,from sharks). As used herein, “human” antibodies include antibodieshaving the amino acid sequence of a human immunoglobulin and includeantibodies isolated from human immunoglobulin libraries or from animalstransgenic for one or more human immunoglobulins and can in someinstances express endogenous immunoglobulins and some not, as describedinfra and, for example in, U.S. Pat. No. 5,939,598 by Kucherlapati etal.

As used herein, the term “heavy chain subunit” includes amino acidsequences derived from an immunoglobulin heavy chain, a bindingmolecule, e.g., an antibody comprising a heavy chain subunit includes atleast one of: a VH domain, a CH1 domain, a hinge (e.g., upper, middle,and/or lower hinge region) domain, a CH2 domain, a CH3 domain, a CH4domain, or a variant or fragment thereof. For example, a bindingmolecule, e.g., an antibody or fragment, variant, or derivative thereofcan include, in addition to a VH domain, a CH1 domain; CH1 domain, ahinge, and a CH2 domain; a CH1 domain and a CH3 domain; a CH1 domain, ahinge, and a CH3 domain; or a CH1 domain, a hinge domain, a CH2 domain,and a CH3 domain. In certain aspects a binding molecule, e.g., anantibody or fragment, variant, or derivative thereof can include, inaddition to a VH domain, a CH3 domain and a CH4 domain; or a CH3 domain,a CH4 domain, and a J chain. Further, a binding molecule for use in thedisclosure can lack certain constant region portions, e.g., all or partof a CH2 domain. It will be understood by one of ordinary skill in theart that these domains (e.g., the heavy chain subunit) can be modifiedsuch that they vary in amino acid sequence from the originalimmunoglobulin molecule.

The heavy chain subunits of a binding molecule, e.g., an antibody orfragment thereof, can include domains derived from differentimmunoglobulin molecules. For example, a heavy chain subunit of apolypeptide can include a CH1 domain derived from an IgG1 molecule and ahinge region derived from an IgG3 molecule. In another example, a heavychain subunit can include a hinge region derived, in part, from an IgG1molecule and, in part, from an IgG3 molecule. In another example, aheavy chain subunit can comprise a chimeric hinge derived, in part, froman IgG1 molecule and, in part, from an IgG4 molecule.

As used herein, the term “light chain subunit” includes amino acidsequences derived from an immunoglobulin light chain. The light chainsubunit includes at least one of a VL or CL (e.g., Cκ or Cλ) domain.

Binding molecules, e.g., antibodies or antigen-binding fragments,variants, or derivatives thereof can be described or specified in termsof the epitope(s) or portion(s) of an antigen that they recognize orspecifically bind. The portion of a target antigen that specificallyinteracts with the antigen binding domain of an antibody is an“epitope,” or an “antigenic determinant.” A target antigen can comprisea single epitope or at least two epitopes, and can include any number ofepitopes, depending on the size, conformation, and type of antigen.

As previously indicated, the subunit structures and three dimensionalconfiguration of the constant regions of the various immunoglobulinclasses are well known. As used herein, the term “VH domain” includesthe amino terminal variable domain of an immunoglobulin heavy chain andthe term “CH1 domain” includes the first (most amino terminal) constantregion domain of an immunoglobulin heavy chain. The CH1 domain isadjacent to the VH domain and is amino terminal to the hinge region of atypical immunoglobulin heavy chain molecule.

As used herein the term “CH2 domain” includes the portion of a heavychain molecule that extends, e.g., from about amino acid 244 to aminoacid 360 of an IgG antibody using conventional numbering schemes (aminoacids 244 to 360, Kabat numbering system; and amino acids 231-340, EUnumbering system; see Kabat E A et al. op. cit. The CH3 domain extendsfrom the CH2 domain to the C-terminal of the IgG molecule and comprisesapproximately 108 amino acids. Certain immunoglobulin classes, e.g.,IgM, further include a CH4 region.

As used herein, the term “hinge region” includes the portion of a heavychain molecule that joins the CH1 domain to the CH2 domain. This hingeregion comprises approximately 25 amino acids and is flexible, thusallowing the two N-terminal antigen binding regions to moveindependently.

As used herein the term “disulfide bond” includes the covalent bondformed between two sulfur atoms. The amino acid cysteine comprises athiol group that can form a disulfide bond or bridge with a second thiolgroup. In certain IgG molecules, the CH1 and CL regions are linked by adisulfide bond and the two heavy chains are linked by two disulfidebonds at positions corresponding to 239 and 242 using the Kabatnumbering system (position 226 or 229, EU numbering system). In certainaspects provided herein, a human IgG4 Fc domain can be mutated in thehinge region to insure disulfide bond formation between two hingeregions, specifically, a serine to proline mutation at position 228(according to EU numbering). Human IgG4 Fc domains comprising the S228Pmutation are referred to herein as “IgG4P Fc domains.”

As used herein, an “Fc-TM region” is a human IgG Fc region thatcomprises amino acid substitutions of L234F, L235E and P331S as numberedby the EU index as set forth in Kabat and exhibits reduced or ablatedeffector (ADCC and/or CDC) function, reduced or ablated binding to Fcreceptors, and/or reduced or ablated toxicities. See, e.g., U.S. PatentApplication Publication No. 2011/0059078, which is incorporated hereinby reference in its entirety.

As used herein, the term “chimeric antibody” refers to an antibody inwhich the immunoreactive region or site is obtained or derived from afirst species and the constant region (which can be intact, partial ormodified) is obtained from a second species. In some embodiments thetarget binding region or site will be from a non-human source (e.g.mouse or primate) and the constant region is human.

The terms “multispecific antibody, or “bispecific antibody” refer to anantibody that has binding domains for two or more different epitopeswithin a single antibody molecule. Other binding molecules in additionto the canonical antibody structure can be constructed with two bindingspecificities. Epitope binding by bispecific or multispecific antibodiescan be simultaneous or sequential. Triomas and hybrid hybridomas are twoexamples of cell lines that can secrete bispecific antibodies.Bispecific antibodies can also be constructed by recombinant means.(Ströhlein and Heiss, Future Oncol. 6:1387-94 (2010); Mabry and Snavely,IDrugs. 13:543-9 (2010)). A bispecific antibody can also be a diabody.

As used herein, the term “engineered antibody” refers to an antibody inwhich the variable domain in either the heavy and light chain or both isaltered by at least partial replacement of one or more amino acids ineither the CDR or framework regions. In certain aspects entire CDRs froman antibody of known specificity can be grafted into the frameworkregions of a heterologous antibody. Although alternate CDRs can bederived from an antibody of the same class or even subclass as theantibody from which the framework regions are derived, CDRs can also bederived from an antibody of different class, e.g., from an antibody froma different species. An engineered antibody in which one or more “donor”CDRs from a non-human antibody of known specificity are grafted into ahuman heavy or light chain framework region is referred to herein as a“humanized antibody.” In certain aspects not all of the CDRs arereplaced with the complete CDRs from the donor variable region and yetthe antigen binding capacity of the donor can still be transferred tothe recipient variable domains. Given the explanations set forth in,e.g., U.S. Pat. Nos. 5,585,089, 5,693,761, 5,693,762, and 6,180,370, itwill be well within the competence of those skilled in the art, eitherby carrying out routine experimentation or by trial and error testing toobtain a functional engineered or humanized antibody.

As used herein the term “engineered” includes manipulation of nucleicacid or polypeptide molecules by synthetic means (e.g. by recombinanttechniques, in vitro peptide synthesis, by enzymatic or chemicalcoupling of peptides or some combination of these techniques).

As used herein, the terms “linked,” “fused” or “fusion” or othergrammatical equivalents can be used interchangeably. These terms referto the joining together of two more elements or components, by whatevermeans including chemical conjugation or recombinant means. An “in-framefusion” refers to the joining of two or more polynucleotide open readingframes (ORFs) to form a continuous longer ORF, in a manner thatmaintains the translational reading frame of the original ORFs. Thus, arecombinant fusion protein is a single protein containing two or moresegments that correspond to polypeptides encoded by the original ORFs(which segments are not normally so joined in nature.) Although thereading frame is thus made continuous throughout the fused segments, thesegments can be physically or spatially separated by, for example,in-frame linker sequence. For example, polynucleotides encoding the CDRsof an immunoglobulin variable region can be fused, in-frame, but beseparated by a polynucleotide encoding at least one immunoglobulinframework region or additional CDR regions, as long as the “fused” CDRsare co-translated as part of a continuous polypeptide.

In the context of polypeptides, a “linear sequence” or a “sequence” isan order of amino acids in a polypeptide in an amino to carboxylterminal direction in which amino acids that neighbor each other in thesequence are contiguous in the primary structure of the polypeptide. Aportion of a polypeptide that is “amino-terminal” or “N-terminal” toanother portion of a polypeptide is that portion that comes earlier inthe sequential polypeptide chain. Similarly a portion of a polypeptidethat is “carboxy-terminal” or “C-terminal” to another portion of apolypeptide is that portion that comes later in the sequentialpolypeptide chain. For example in a typical antibody, the variabledomain is “N-terminal” to the constant region, and the constant regionis “C-terminal” to the variable domain.

The term “expression” as used herein refers to a process by which a geneproduces a biochemical, for example, a polypeptide. The process includesany manifestation of the functional presence of the gene within the cellincluding, without limitation, gene knockdown as well as both transientexpression and stable expression. It includes without limitationtranscription of the gene into messenger RNA (mRNA), and the translationof such mRNA into polypeptide(s). If the final desired product is abiochemical, expression includes the creation of that biochemical andany precursors. Expression of a gene produces a “gene product.” As usedherein, a gene product can be either a nucleic acid, e.g., a messengerRNA produced by transcription of a gene, or a polypeptide which istranslated from a transcript. Gene products described herein furtherinclude nucleic acids with post transcriptional modifications, e.g.,polyadenylation, or polypeptides with post translational modifications,e.g., methylation, glycosylation, the addition of lipids, associationwith other protein subunits, proteolytic cleavage, and the like.

Terms such as “treating” or “treatment” or “to treat” or “alleviating”or “to alleviate” refer to therapeutic measures that cure, slow down,lessen symptoms of, and/or halt or slow the progression of an existingdiagnosed pathologic condition or disorder. Terms such as “prevent,”“prevention,” “avoid,” “deterrence” and the like refer to prophylacticor preventative measures that prevent the development of an undiagnosedtargeted pathologic condition or disorder. Thus, “those in need oftreatment” can include those already with the disorder; those prone tohave the disorder; and those in whom the disorder is to be prevented.

OX40, or “OX40 receptor” is a protein (also variously termed CD134,tumor necrosis factor receptor superfamily member 4, and ACT-35)expressed on the surface of activated T cells, e.g., CD4⁺ and CD8⁺ Tcells, as well as on Foxp3⁺CD4⁺ regulatory T cells (Tregs). Naive CD4⁺and CD8⁺ T cells do not express OX40 (Croft, M., (2010) Ann Rev Immunol28:57-78).

“OX40 ligand” (“OX40L”) (also variously termed tumor necrosis factorligand superfamily member 4, gp34, TAX transcriptionally-activatedglycoprotein-1, and CD252) is found largely on antigen presenting cells(APCs), and can be induced on activated B cells, dendritic cells (DCs),Langerhans cells, plamacytoid DCs, and macrophages (Id.). Other cells,including activated T cells, NK cells, mast cells, endothelial cells,and smooth muscle cells can express OX40L in response to inflammatorycytokines (Id.). OX40L specifically binds to the OX40 receptor. Thehuman protein is described in PCT Publication No. WO 95/21915. The mouseOX40L is described in U.S. Pat. No. 5,457,035. OX40L is expressed on thesurface of cells and includes an intracellular, a transmembrane and anextracellular receptor-binding domain. A functionally active solubleform of OX40L can be produced by deleting the intracellular andtransmembrane domains as described, e.g., in U.S. Pat. Nos. 5,457,035and 6,312,700, and WO 95/21915, the disclosures of which areincorporated herein for all purposes. A functionally active form ofOX40L is a form that retains the capacity to bind specifically to OX40,that is, that possesses an OX40 “receptor binding domain.” Methods ofdetermining the ability of an OX40L molecule or derivative to bindspecifically to OX40 are discussed below. Methods of making and usingOX40L and its derivatives (such as derivatives that include an OX40binding domain) are described in WO 95/21915, which also describesproteins comprising the soluble form of OX40L linked to other peptides,such as human immunoglobulin (“Ig”) Fc regions, that can be produced tofacilitate purification of OX40 ligand from cultured cells, or toenhance the stability of the molecule after in vivo administration to amammal (see also, U.S. Pat. No. 5,457,035 and PCT Publication No. WP2006/121810, both of which are incorporated by reference herein in theirentireties).

By “subject” or “individual” or “animal” or “patient” or “mammal,” ismeant any subject, particularly a mammalian subject, for whom diagnosis,prognosis, or therapy is desired. Mammalian subjects include humans,domestic animals, farm animals, and zoo, sports, or pet animals such asdogs, cats, guinea pigs, rabbits, rats, mice, horses, swine, cows,bears, and so on.

As used herein, phrases such as “a subject that would benefit fromtherapy” and “an animal in need of treatment” includes subjects, such asmammalian subjects, that would benefit from administration of ahumanized anti-OX40 antibody. Such antibodies, can be used, e.g., for adiagnostic procedures and/or for treatment or prevention of a disease,e.g., cancer.

Humanized Anti-OX40 Antibodies and Antigen-Binding Fragments Thereof

The present disclosure relates to antibodies, e.g., humanizedantibodies, which specifically bind to OX40. In certain aspects, anantibody or fragment thereof as provided herein can be isolated. Incertain aspects, an antibody or fragment thereof as provided herein canbe substantially pure. In certain aspects an antibody or fragmentthereof as provided herein can be non-naturally occurring.

In certain aspects, this disclosure provides a humanized anti-OX40antibody or an antigen-binding fragment thereof comprising the VH and VLof OX40mAb5, OX40mAb8, OX40mAb10, OX40mAb11, OX40mAb12, OX40mAb13,OX40mAb14, OX40mAb15, OX40mAb16, OX40mAb17, OX40mAb18, OX40mAb19,OX40mAb20, OX40mAb21, OX40mAb22, OX40mAb23, OX40mAb24, OX40mAb25,OX40mAb25a, OX40mAb26, OX40mAb27, OX40mAb28, OX40mAb29, OX40mAb30,OX40mAb31, OX40mAb32, or OX40mAb37. In certain aspects, this disclosureprovides a humanized anti-OX40 antibody or an antigen-binding fragmentthereof comprising the heavy chain and light chain of OX40mAb5,OX40mAb8, OX40mAb10, OX40mAb11, OX40mAb12, OX40mAb13, OX40mAb14,OX40mAb15, OX40mAb16, OX40mAb17, OX40mAb18, OX40mAb19, OX40mAb20,OX40mAb21, OX40mAb22, OX40mAb23, OX40mAb24, OX40mAb25, OX40mAb25a,OX40mAb26, OX40mAb27, OX40mAb28, OX40mAb29, OX40mAb30, OX40mAb31, orOX40mAb32.

In certain aspects this disclosure provides a humanized anti-OX40antibody or an antigen-binding fragment thereof comprising an antibodyVH and an antibody VL, wherein the VL comprises an amino acid sequenceat least 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to thereference amino acid sequence SEQ ID NO: 29 or SEQ ID NO: 32.

In certain aspects this disclosure provides a humanized anti-OX40antibody or an antigen-binding fragment thereof comprising an antibodyVH and an antibody VL, where the VL comprises SEQ ID NO: 29 or SEQ IDNO: 32.

The disclosure further provides a humanized anti-OX40 antibody or anantigen-binding fragment thereof comprising an antibody VH and anantibody VL, wherein the VH comprises VH-CDR1, VH-CDR2, and VH-CDR3amino acid sequences identical to, or identical except for eight, seven,six, five, four, three, two, or one single amino acid substitutions,deletions, or insertions in one or more of the VH-CDRS to: the VHCDR1amino acid sequence SEQ ID NO: 8, the VHCDR2 amino acid sequence SEQ IDNO: 14, SEQ ID NO: 15, or SEQ ID NO: 16, and the VHCDR3 amino acidsequence SEQ ID NO: 25, SEQ ID NO: 26, or SEQ ID NO: 27.

The disclosure further provides a humanized anti-OX40 antibody or anantigen-binding fragment thereof comprising an antibody VH and anantibody VL, wherein the VH comprises an amino acid sequence with theformula:HFW1-HCDR1-HFW2-HCDR2-HFW3-HCDR3-HFW4,wherein HFW1 is SEQ ID NO: 6 or SEQ ID NO: 7, HCDR1 is SEQ ID NO: 8,HFW2 is SEQ ID NO: 9, HCDR2 is SEQ ID NO: 14, SEQ ID NO: 15 or SEQ IDNO: 16, HFW3 is SEQ ID NO: 17, HCDR3 is SEQ ID NO: 25, SEQ ID NO: 26, orSEQ ID NO: 27, and HFW4 is SEQ ID NO: 28. In certain aspects the aminoacid sequence of HFW2 is SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, orSEQ ID NO: 13. In certain aspects the amino acid sequence of HFW3 is SEQID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22,SEQ ID NO: 23, or SEQ ID NO: 24.

Moreover, the disclosure provides a humanized anti-OX40 antibody or anantigen-binding fragment thereof comprising an antibody VH and anantibody VL, wherein the VH comprises an amino acid sequence at least70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to the reference aminoacid sequence SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO:39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ IDNO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, or SEQ ID NO:67.

In one aspect, the disclosure provides a humanized anti-OX40 antibody oran antigen-binding fragment thereof comprising an antibody VH and anantibody VL, where the VL comprises the amino acid sequence SEQ ID NO:29 and the VH comprises the amino acid sequence SEQ ID NO: 59.

A humanized anti-OX40 antibody or an antigen-binding fragment thereof asprovided herein can include, in addition to a VH and a VL, a heavy chainconstant region or fragment thereof. In certain aspects the heavy chainconstant region is a human heavy chain constant region, e.g., a humanIgG constant region, e.g., a human IgG1 constant region or a human IgG4constant region. As described elsewhere herein, in certain aspects aheavy chain constant region or fragment thereof, e.g., a human IgGconstant region or fragment thereof, can include one or more amino acidsubstitutions relative to a wild-type IgG constant region, where themodified IgG has one or more desirable properties relative to awild-type IgG constant region. For example, the human IgG constantregion can be an IgG4P constant region, or an IgG1-TM constant region,as described elsewhere herein.

A humanized anti-OX40 antibody or an antigen-binding fragment thereof asprovided herein can include, in addition to a VH and a VL, andoptionally a heavy chain constant region or fragment thereof, a lightchain constant region or fragment thereof. In certain aspects the lightchain constant region is a kappa or lambda light chain constant region,e.g., a human kappa constant region or a human lambda constant region.In a specific aspect, the light chain constant region is a human kappaconstant region.

In certain aspects the disclosure provides a humanized anti-OX40antibody or an antigen-binding fragment thereof comprising an antibodyheavy chain or fragment thereof and an antibody light chain or fragmentthereof, where the heavy chain comprises the amino acid sequence SEQ IDNO: 71, and the light chain comprises the amino acid sequence SEQ ID NO:30.

A humanized anti-OX40 antibody or an antigen-binding fragment thereof asprovided herein can be, e.g., monoclonal, polyclonal, recombinant,multispecific, or any combination thereof. A humanized anti-OX40antibody or antigen-binding fragment can be, e.g., an Fv fragment, anFab fragment, an F(ab′)2 fragment, an Fab′ fragment, a dsFv fragment, anscFv fragment, an sc(Fv)2 fragment, an Fd fragment, a disulfide linkedFv fragment, a V-NAR domain, an IgNar, an intrabody, an IgGACH2, aminibody, a F(ab′)3 fragment, a tetrabody, a triabody, a diabody, asingle-domain antibody, a DVD-Ig, a Fcab fragment, an mAb2 fragment, an(scFv)2 fragment, or a scFv-Fc fragment.

In certain aspects, a humanized anti-OX40 antibody or an antigen-bindingfragment thereof as provided herein can specifically bind to human,cynomolgus monkey, or rhesus monkey OX40. In certain aspects, ahumanized anti-OX40 antibody or an antigen-binding fragment thereof asprovided herein can specifically bind to the surface of primary human,cyno, or rhesus CD4 T cells or Jurkat cells. In certain aspects, ahumanized anti-OX40 antibody or an antigen-binding fragment thereof asprovided herein can specifically bind to OX40 as expressed on activatedCD4⁺ or CD8⁺ T cells from human, cynomolgus monkey, rhesus monkey, orany combination thereof. In certain aspects, a humanized anti-OX40antibody or an antigen-binding fragment thereof as provided herein canspecifically bind to OX40 as expressed on primary activated CD4⁺ T cellsfrom human, cynomolgus monkey, rhesus monkey, or any combinationthereof. In certain aspects, a humanized anti-OX40 antibody or anantigen-binding fragment thereof as provided herein does not bind tomurine or rat OX40. In certain aspects, a humanized anti-OX40 antibodyor an antigen-binding fragment thereof as provided herein does not crossreact with related TNFRSF proteins, e.g., the TNFRSF proteins listed inTable 3-1 in Example 3 below.

A humanized anti-OX40 antibody or antigen-binding fragment thereof asprovided herein can contain one or more conservative amino acid changes,e.g., up to ten conservative changes (e.g., two substituted amino acids,three substituted amino acids, four substituted amino acids, or fivesubstituted amino acids, etc.), provided that the changes can be made inthe polypeptide without changing a biochemical function of the humanizedanti-OX40 antibody or antigen-binding fragment thereof, e.g.,specifically binding to OX40, thereby triggering signaling. For example,one or more conservative changes can be made in a receptor bindingdomain of an antibody as provided herein without blocking its ability tobind to OX40.

Additionally, part of a polypeptide domain can be deleted withoutimpairing or eliminating all of its functions. Similarly, insertions oradditions can be made in the polypeptide chain, for example, addingepitope tags, without impairing or eliminating its functions, asdescribed below. Other modifications that can be made without materiallyimpairing one or more functions of a polypeptide include, for example,in vivo or in vitro chemical and biochemical modifications thatincorporate unusual amino acids. Such modifications include, forexample, acetylation, carboxylation, phosphorylation, glycosylation,labeling, e.g., with radionuclides, and various enzymatic modifications,as will be readily appreciated by those of ordinary skill in the art. Avariety of methods for labeling polypeptides, and labels useful for suchpurposes, are well known in the art, and include radioactive isotopessuch as ³²P, fluorophores, chemiluminescent agents, enzymes, andantiligands.

A humanized anti-OX40 antibody or antigen-binding fragment thereof asprovided herein can further include a heterologous agent, e.g., astabilizing agent, an immune response modifier, or a detectable agent.In certain aspects the heterologous agent comprises one or moreadditional polypeptide sequences fused to the polypeptide subunit via apeptide bond, such as a signal sequence (e.g., a secretory signalsequence), a linker sequence, an amino acid tag or label, or a peptideor polypeptide sequence that facilitates purification. In certainaspects, the heterologous polypeptide can be fused to the N-terminus orthe C-terminus of either a heavy chain or light chain antibody subunit,or fragment thereof, as long as the functional characteristics of thedomains are maintained.

In certain aspects, the heterologous agent can be chemically conjugatedto a humanized anti-OX40 antibody or antigen-binding fragment thereof asprovided herein. Exemplary heterologous agents that can be chemicallyconjugated to the polypeptide subunit include, without limitation,linkers, drugs, toxins, imaging agents, radioactive compounds, organicand inorganic polymers, and any other compositions which can provide adesired activity that is not provided by the polypeptide subunit itself.Specific agents include, without limitation, polyethylene glycol (PEG),a cytotoxic agent, a radionuclide, an imaging agent, biotin.

In certain aspects, the disclosure provides certain anti-OX40 antibodiesto be used as controls or research tools. For example, the disclosureprovides a humanized anti-OX40 antibody or antigen-binding fragmentthereof as described above, in which the VH and VL are fused to a murineIgG1 heavy chain and a murine kappa light chain, respectively. This“reverse chimera” is useful for in vivo characterization in rhesusmonkeys. In another example, the VH region on a humanized anti-OX40antibody as provided herein can be attached to various heavy chainconstant regions with altered effector functions, e.g., human IgG4P orhuman IgG1TM, described elsewhere herein.

A humanized anti-OX40 antibody or antigen-binding fragment thereof asprovided herein can specifically bind to OX40 as expressed on primaryactivated T cells, e.g., primary activated CD4⁺ T cells, primaryactivated CD8⁺ T cells and/or regulatory T cells, from human, cynomolgusmonkey, rhesus monkey, or any combination thereof. In certain aspects, ahumanized anti-OX40 antibody or antigen-binding fragment thereof asprovided herein, e.g., OX40mAb24, can bind to human OX40 expressed onprimary activated human CD4⁺ T cells with a binding affinity of about0.01 pM to about 1 nM, e.g., about 1 pM to about 500 pM, e.g., about 100pM to about 400 pM, e.g., about 250 pM to about 370 pM, all as measuredby flow cytometry. For example, a humanized anti-OX40 antibody or anantigen-binding fragment thereof can bind to human OX40 expressed onprimary activated human CD4⁺ T cells with a binding affinity of about0.1 pM, about 0.5 pM, about 1 pM, about 10 pM, about 50 pM, about 100pM, about 150 pM, about 200 pM, about 250 pM, about 275 pM, about 300pM, about 325 pM, about 350 pM, about 370 pM, about 400 pM, about 425pM, about 450 pM, about 475 pM, about 500 pM, about 550 pM, about 600pM, about 650 pM, about 700 pM, about 750 pM, or about 1 nM, all asmeasured by flow cytometry. In certain aspects, a humanized anti-OX40antibody or antigen-binding fragment thereof as provided herein can bindto human OX40 expressed on primary activated human CD4⁺ T cells with abinding affinity of about 312 pM. Binding affinity can be measured by anumber of different methods and/or instruments, and the relative bindingaffinities can vary depending on the method or instrument, as is wellunderstood by persons or ordinary skill in the art.

A humanized anti-OX40 antibody or antigen-binding fragment thereof asprovided herein can occupy and cross-link some or all of the OX40molecules on the surface of a cell, e.g., a primary activated human CD4⁺T cell. In certain aspects, a humanized anti-OX40 antibody orantigen-binding fragment thereof as provided herein can bind to humanOX40 expressed on primary activated human CD4⁺ T cells, and can achieve20% receptor occupancy on primary activated human CD4⁺ T cells (EC₂₀) ata concentration of about 0.01 pM to about 1 nM, e.g., about 0.1 pM toabout 500 pM, e.g., about 1 pM to about 200 pM, e.g., about 10 pM toabout 100 pM, e.g., about 50 pM to about 100 pM, e.g., about 63 pM toabout 93 pM, e.g., about 1 pM, about 10 pM, about 20 pM, about 30 pM,about 40 pM, about 50 pM, about 60 pM, about 70 pM, about 78 pM, about80 pM, about 90 pM, about 100 pM, or about 150 pM, as measured by flowcytometry. In certain aspects, a humanized anti-OX40 antibody orantigen-binding fragment thereof as provided herein can achieve 20%receptor occupancy on primary activated human CD4⁺ T cells (EC₂₀) at aconcentration of about 78 pM., as measured by flow cytometry. In certainaspects, a humanized anti-OX40 antibody or antigen-binding fragmentthereof as provided herein can bind to human OX40 expressed on primaryactivated human CD4⁺ T cells, and can achieve 50% receptor occupancy onprimary activated human CD4⁺ T cells (EC₅₀) at a concentration of about0.01 pM to about 1 nM, e.g., about 1 pM to about 500 pM, e.g., about 100pM to about 400 pM, e.g., about 250 pM to about 370 pM, e.g., about 100pM, about 150 pM, about 200 pM, about 250 pM, about 275 pM, about 300pM, about 325 pM, about 350 pM, about 370 pM, about 400 pM, about 425pM, about 450 pM, about 475 pM, or about 500 pM, all as measured by flowcytometry. In certain aspects, a humanized anti-OX40 antibody orantigen-binding fragment thereof as provided herein can achieve 50%receptor occupancy on primary activated human CD4⁺ T cells (EC₅₀) at aconcentration of about 312 pM., as measured by flow cytometry. Incertain aspects, a humanized anti-OX40 antibody or an antigen-bindingfragment thereof can bind to human OX40 expressed on primary activatedhuman CD4⁺ T cells, and can achieve 90% receptor occupancy on primaryactivated human CD4⁺ T cells (EC₉₀) at a concentration of about 100 pMto about 100 nM, e.g., about 500 pM to about 10 nM, e.g., about 1 nM toabout 500 nM, e.g., about 2 nM to about 4 nM, e.g., about 1 nM, about 2nM, about 3 nM, about 4 nM, or about 5 nM, as measured by flowcytometry. In certain aspects, a humanized anti-OX40 antibody orantigen-binding fragment thereof as provided herein can achieve 90%receptor occupancy on primary activated human CD4⁺ T cells (EC₉₀) at aconcentration of about 2290 pM to about 3330 pM, e.g., about 2810 pM.,as measured by flow cytometry.

In certain aspects, a humanized anti-OX40 antibody or antigen-bindingfragment thereof as provided herein can bind to human OX40 expressed onOX40-overexpressing Jurkat cells with a binding affinity of about 250 pMto about 600 pM, e.g., about 424 pM as measured by flow cytometry.

In certain aspects, a humanized anti-OX40 antibody or an antigen-bindingfragment thereof can bind to human OX40 expressed on OX40-overexpressingJurkat cells, and can achieve EC₂₀ at a concentration of about 60 toabout 150 pM, EC₅₀ at a concentration of about 250 to about 600 pM, andEC₉₀ at a concentration about 2260 to about 4390 pM as measured by flowcytometry. In certain aspects, a humanized anti-OX40 antibody orantigen-binding fragment thereof as provided herein can bind to humanOX40 expressed on OX40-overexpressing Jurkat cells and can achieve EC₂₀at a concentration of about 106 pM, EC₅₀ at a concentration of about 424pM, and EC₉₀ at a concentration of about 3820 pM, as measured by flowcytometry.

In another example, a humanized anti-OX40 antibody or antigen-bindingfragment thereof as provided herein can bind to cynomolgus monkey OX40expressed on primary activated cynomolgus monkey CD4⁺ T cells with abinding affinity of about 340 pM to about 820 pM, e.g., about 580 pM, asmeasured by flow cytometry. In another example, a humanized anti-OX40antibody or antigen-binding fragment thereof as provided herein can bindto rhesus monkey OX40 expressed on primary activated rhesus monkey CD4⁺T cells with a binding affinity of about 130 pM to about 600 pM, e.g.,about 370 pM, as measured by flow cytometry.

In certain aspects, a humanized anti-OX40 antibody or antigen-bindingfragment thereof as provided herein can induce dose-dependentproliferation of activated CD4⁺ T cells in a plate-based assay. Forexample, in an in vitro assay a humanized anti-OX40 antibody orantigen-binding fragment thereof as provided herein, e.g., OX40mAb24, a20% maximal proliferation response (EC₂₀) can be achieved in primaryactivated human CD4⁺ T cells at an antibody concentration of about 14 pMto about 28 pM, e.g., about 21 pM, a 50% maximal proliferation response(EC₅₀) can be achieved in primary activated human CD4⁺ T cells at anantibody concentration of about 0.3 pM to about 130 pM, e.g., about 28pM, and a 90% maximal proliferation response (EC₉₀) can be achieved inprimary activated human CD4⁺ T cells at an antibody concentration ofabout 50 pM to about 90 pM, e.g., about 72 pM, all as measured by flowcytometry.

In certain aspects, a humanized anti-OX40 antibody or antigen-bindingfragment thereof as provided herein can induce dose-dependent cytokinerelease from activated CD4⁺ T cells, e.g., human primary activated CD4⁺T cells. In certain aspects, the released cytokine is IFNγ, TNFα, IL-5,IL-10, IL-2, IL-4, IL-13, IL-8, IL-12 p70, IL-1β, or any combinationthereof. In certain aspects, the cytokine is IFNγ, TNFα, IL-5, IL-10,IL-13 or any combination thereof. Similarly, a humanized anti-OX40antibody or antigen-binding fragment thereof as provided herein canachieve CD4⁺ T cell proliferation and cytokine release in primaryactivated cynomolgus monkey CD4⁺ T cells and in primary activated rhesusmonkey CD4⁺ T cells.

In additional aspects, a humanized anti-OX40 antibody or antigen-bindingfragment thereof as provided herein can activate the NFκB pathway inOX40 expressing T cells in the presence of FcγR-expressing cells. Forexample, a humanized anti-OX40 antibody or antigen-binding fragmentthereof as provided herein can activate the NFκB pathway inOX40-expressing Jurkat NFκB-luciferase reporter cells that produceluciferase in response to stimulation of the NFκB signaling pathway.Alternatively, a humanized anti-OX40 antibody or antigen-bindingfragment thereof as provided herein can activate the NFκB pathway incells expressing human OX40, cynomolgus monkey OX40 or rhesus monkeyOX40.

In yet another aspect a humanized anti-OX40 antibody or antigen-bindingfragment thereof as provided herein can facilitate cancer treatment,e.g., by slowing tumor growth, stalling tumor growth, or reducing thesize of existing tumors, when administrated as an effective dose to asubject in need of cancer treatment. In certain aspects the facilitationof cancer treatment can be achieved in the presence of T cells. Incertain aspects, a humanized anti-OX40 antibody or antigen-bindingfragment thereof as provided herein, when administered as an effectivedose to a subject in need of treatment, can reduce tumor growth by atleast 10%, at least 20%, at least 30%, at least 40%, and least 50%, atleast 60%, or at least 70% compared to administration of anisotype-matched control antibody.

In yet further aspects, a humanized anti-OX40 antibody orantigen-binding fragment thereof as provided herein can induceproliferation of activated, OX40-expressing T cells through binding toOX40, and at the same time trigger complement-dependent orantibody-dependent cellular cytotoxicity against the OX40-expressing Tcells, e.g., activated CD4⁺ T cells, activated CD8⁺ T cells and/orregulatory T cells. Moreover in certain aspects, a humanized anti-OX40antibody or antigen-binding fragment thereof as provided herein caninduce proliferation of OX40-expressing T cells, e.g., activated,OX40-expressing CD4⁺, CD8⁺ T cells and/or regulatory T cells throughbinding to OX40, and at the same time bind to C1q or trigger NKcell-mediated antibody-dependent cellular cytotoxicity ofOX40-expressing T cells, e.g., activated CD4⁺ T cells, activated CD8+ Tcells and/or regulatory T cells.

In certain aspects the disclosure provides an anti-OX40 antibody orfragment thereof comprising a humanized VH attached to a murine heavychain constant region and a humanized VL attached to a murine lightchain constant region, wherein the heavy chain comprises SEQ ID NO: 81and the light chain comprises SEQ ID NO: 83. In certain aspects theheavy chain constant region can be, e.g., a murine IgG1 constant region.In certain aspects the light chain constant region can be, e.g., amurine kappa constant region.

In certain aspects the disclosure provides a rat-anti-mouse OX40antibody or antigen-binding fragment thereof comprising a rat VH and arat VL, where the VH comprises the amino acid sequence SEQ ID NO: 85 andthe VL comprises the amino acid sequence SEQ ID NO: 88. In certainaspects, this antibody or fragment thereof further comprises a lightchain constant region or fragment thereof fused to the C-terminus of theVL. The light chain constant region can be, e.g., a murine kappaconstant region. In certain aspects, this antibody or fragment thereoffurther comprises a heavy chain constant region or fragment thereoffused to the C-terminus of the VH. The heavy chain constant region canbe, e.g., a murine IgG2a constant region. In certain aspects thisrat-anti-mouse OX40 antibody comprises the heavy chain amino acidsequence SEQ ID NO: 86 and the light chain amino acid sequence SEQ IDNO: 89. In certain aspects, a rat-anti-mouse OX40 antibody or fragmentthereof as provided herein can specifically bind to mouse OX40. Incertain aspects, administration of an effective dose of a rat-anti-mouseOX40 antibody or fragment thereof as provided herein to mouse caninhibit mouse cancer cell line growth in the mouse.

In certain aspects a rat-anti-mouse OX40 antibody fragment as providedherein can be, e.g., an Fv fragment, an Fab fragment, an F(ab′)2fragment, an Fab′ fragment, a dsFv fragment, an scFv fragment, or ansc(Fv)2 fragment, or any combination thereof.

Polynucleotides Encoding Humanized Anti-OX40 Antibodies, Fragments, orSubunits

This disclosure provides polynucleotides comprising nucleic acidsequences that encode a humanized anti-OX40 antibody or anantigen-binding fragment thereof. For example, the disclosure provides apolynucleotide, or two or more polynucleotides, comprising a nucleicacid sequence that encodes a humanized anti-OX40 antibody or a subunitof a humanized anti-OX40 antibody, or encodes an antigen-bindingfragment of such an antibody. The polynucleotides of the disclosure canbe in the form of RNA or in the form of DNA. DNA includes cDNA, genomicDNA, e.g., modified genomic DNA, and synthetic DNA; and can bedouble-stranded or single-stranded, and if single stranded can be thecoding strand or non-coding (anti-sense) strand.

In certain aspects, a polynucleotide can be isolated. In certainaspects, a polynucleotide can be substantially pure. In certain aspectsa polynucleotide can be non-naturally occurring. In certain aspects apolynucleotide can be cDNA or derived from cDNA. In certain aspects apolynucleotide can be recombinantly produced. In certain aspects apolynucleotide can comprise the coding sequence for the maturepolypeptide fused in the same reading frame to a polynucleotide whichaids, for example, in expression and secretion of a polypeptide from ahost cell (e.g., a leader sequence which functions as a secretorysequence for controlling transport of a polypeptide from the cell). Thepolypeptide having a leader sequence is a preprotein and can have theleader sequence cleaved by the host cell to form the mature form of thepolypeptide.

In certain aspects the disclosure provides a polynucleotide comprising anucleic acid that encodes a humanized anti-OX40 antibody or anantigen-binding fragment thereof as provided herein, or a polypeptidesubunit of a humanized anti-OX40 antibody or an antigen-binding fragmentthereof as provided herein.

Also provided are polynucleotides comprising nucleic acid sequencescomprising one or a small number of deletions, additions and/orsubstitutions. Such changes can be contiguous or can be distributed atdifferent positions in the nucleic acid. A substantially identicalnucleic acid sequence can, for example, have 1, or 2, or 3, or 4, oreven more nucleotide deletions, additions and/or substitutions. Incertain aspects, the one or more deletions, additions and/orsubstitutions do not alter the reading frame encoded by thepolynucleotide sequence, such that a modified (“mutant”) butsubstantially identical polypeptide is produced upon expression of thenucleic acid.

In certain aspects this disclosure provides an isolated polynucleotidecomprising a nucleic acid encoding an antibody VL, wherein the VLcomprises an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%,or 100% identical to the reference amino acid sequence SEQ ID NO: 29 orSEQ ID NO: 32. In certain aspects, the polynucleotide encodes anantibody light chain and comprises a nucleotide sequence at least 70%,75%, 80%, 85%, 90%, 95%, or 100% identical to the reference nucleic acidsequence SEQ ID NO: 31.

In certain aspects this disclosure provides an isolated polynucleotidecomprising a nucleic acid encoding an antibody VL, wherein the VLcomprises SEQ ID NO: 29 or SEQ ID NO: 32.

The disclosure further provides an isolated polynucleotide comprising anucleic acid encoding an antibody VH, wherein the VH comprises VH-CDR1,VH-CDR2, and VH-CDR3 amino acid sequences identical to, or identicalexcept for eight, seven, six, five, four, three, two, or one singleamino acid substitutions, deletions, or insertions in one or more of theVH-CDRS to: the VHCDR1 amino acid sequence SEQ ID NO: 8, the VHCDR2amino acid sequence SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16, andthe VHCDR3 amino acid sequence SEQ ID NO: 25, SEQ ID NO: 26, or SEQ IDNO: 27.

The disclosure further provides an isolated polynucleotide comprising anucleic acid encoding an antibody VH, wherein the VH comprises an aminoacid sequence with the formula:HFW1-HCDR1-HFW2-HCDR2-HFW3-HCDR3-HFW4,wherein HFW1 is SEQ ID NO: 6 or SEQ ID NO: 7, HCDR1 is SEQ ID NO: 8,HFW2 is SEQ ID NO: 9, HCDR2 is SEQ ID NO: 14, SEQ ID NO: 15 or SEQ IDNO: 16, HFW3 is SEQ ID NO: 17, HCDR3 is SEQ ID NO: 25, SEQ ID NO: 26, orSEQ ID NO: 27, and HFW4 is SEQ ID NO: 28. In certain aspects the aminoacid sequence of HFW2 is SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, orSEQ ID NO: 13. In certain aspects the amino acid sequence of HFW3 is SEQID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22,SEQ ID NO: 23, or SEQ ID NO: 24.

Moreover, the disclosure provides an isolated polynucleotide comprisinga nucleic acid encoding an antibody VH, wherein the VH comprises anamino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, or 100%identical to the reference amino acid sequence SEQ ID NO: 33, SEQ ID NO:35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ IDNO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63,SEQ ID NO: 65, or SEQ ID NO: 67. In certain aspects, the polynucleotidecomprises a nucleotide sequence at least 70%, 75%, 80%, 85%, 90%, 95%,or 100% identical to the reference nucleic acid sequence SEQ ID NO: 34,SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO:44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ IDNO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQID NO: 64, SEQ ID NO: 66, or SEQ ID NO: 68.

In certain aspects the disclosure provides a polynucleotide comprisingthe nucleic acid of SEQ ID NO: 60, the nucleic acid of SEQ ID NO: 31,the nucleic acid of SEQ ID NO: 72, or any combination thereof.

Further provided is a vector comprising a polynucleotide as describedabove. Suitable vectors are described elsewhere herein, and are known tothose of ordinary skill in the art.

In certain aspects, the disclosure provides a composition, e.g., apharmaceutical composition, comprising a polynucleotide or vector asdescribed above, optionally further comprising one or more carriers,diluents, excipients, or other additives.

In certain aspects, the disclosure provides a polynucleotide compositioncomprising: a polynucleotide that comprises a nucleic acid encoding aVH, and polynucleotide that comprises a nucleic acid encoding a VL.According to this aspect, the VL and VH together can comprise a VL aminoacid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% identicalto the reference amino acid sequence SEQ ID NO: 29 or SEQ ID NO: 32, anda VH comprising: (a) VH-CDR1, VH-CDR2, and VH-CDR3 amino acid sequencesidentical to, or identical except for eight, seven, six, five, four,three, two, or one single amino acid substitutions, deletions, orinsertions in one or more of the VH-CDRS to: the VHCDR1 amino acidsequence SEQ ID NO: 8, the VHCDR2 amino acid sequence SEQ ID NO: 14, SEQID NO: 15, or SEQ ID NO: 16, and the VHCDR3 amino acid sequence SEQ IDNO: 25, SEQ ID NO: 26, or SEQ ID NO: 27; (b) an amino acid sequence withthe formula:HFW1-HCDR1-HFW2-HCDR2-HFW3-HCDR3-HFW4,wherein HFW1 is SEQ ID NO: 6 or SEQ ID NO: 7, HCDR1 is SEQ ID NO: 8,HFW2 is SEQ ID NO: 9, HCDR2 is SEQ ID NO: 14, SEQ ID NO: 15 or SEQ IDNO: 16, HFW3 is SEQ ID NO: 17, HCDR3 is SEQ ID NO: 25, SEQ ID NO: 26, orSEQ ID NO: 27, and HFW4 is SEQ ID NO: 28; or (c) an amino acid sequenceat least 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to thereference amino acid sequence SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO:37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ IDNO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65,or SEQ ID NO: 67. For VH (b), the amino acid sequence of HFW2 can be SEQID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13; and/or theamino acid sequence of HFW3 can be SEQ ID NO: 18, SEQ ID NO: 19, SEQ IDNO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24.

In a polynucleotide composition as described above, the polynucleotidecomprising a nucleic acid encoding a VH and the polynucleotidecomprising a nucleic acid encoding a VL can reside in a single vector,or can be on separate, non-identical vectors. Accordingly the disclosureprovides one or more vectors comprising the polynucleotide compositiondescribed above.

In some cases, a polynucleotide composition encoding a VH and VL asdescribed above can encode a humanized antibody or antigen-bindingfragment thereof that can specifically bind to OX40, e.g., human OX40,cynomolgus monkey OX40, and/or rhesus monkey OX40.

In certain aspects the disclosure provides an isolated polynucleotidecomprising a nucleic acid that encodes an anti-OX40 antibody or fragmentthereof comprising a humanized VH attached to a murine heavy chainconstant region and a humanized VL attached to a murine light chainconstant region, wherein the heavy chain comprises SEQ ID NO: 81 and thelight chain comprises SEQ ID NO: 83. In certain aspects the heavy chainconstant region can be, e.g., a murine IgG1 constant region. In certainaspects the light chain constant region can be, e.g., a murine kappaconstant region.

In certain aspects the disclosure provides an isolated polynucleotidecomprising a nucleic acid that encodes a rat-anti-mouse OX40 antibody orantigen-binding fragment thereof comprising a rat VH and a rat VL, wherethe VH comprises the amino acid sequence SEQ ID NO: 85 and the VLcomprises the amino acid sequence SEQ ID NO: 88. In certain aspects,this antibody or fragment thereof further comprises a light chainconstant region or fragment thereof fused to the C-terminus of the VL.The light chain constant region can be, e.g., a murine kappa constantregion. In certain aspects, this antibody or fragment thereof furthercomprises a heavy chain constant region or fragment thereof fused to theC-terminus of the VH. The heavy chain constant region can be, e.g., amurine IgG2a constant region. In certain aspects this rat-anti-mouseOX40 antibody comprises the heavy chain amino acid sequence SEQ ID NO:86 and the light chain amino acid sequence SEQ ID NO: 89. In certainaspects, a rat-anti-mouse OX40 antibody or fragment thereof as providedherein can specifically bind to mouse OX40. In certain aspects,administration of an effective dose of a rat-anti-mouse OX40 antibody orfragment thereof as provided herein to mouse can inhibit mouse cancercell line growth in the mouse.

This disclosure further provides a host cell comprising apolynucleotide, polynucleotide composition, or vector as provided above,where the host cell can, in some instances, express an antibody orantigen-binding fragment thereof that specifically binds to OX40, e.g.,human OX40, cynomolgus monkey OX40, or rhesus monkey OX40. Such a hostcell can be utilized in a method of making an antibody orantigen-binding fragment thereof as provided herein, which methodincludes (a) culturing the host cell and (b) isolating the antibody orantigen-binding fragment thereof expressed from the host cell.

Polynucleotide variants are also provided. Polynucleotide variants cancontain alterations in the coding regions, non-coding regions, or both.In some aspects polynucleotide variants contain alterations that producesilent substitutions, additions, or deletions, but do not alter theproperties or activities of the encoded polypeptide. In some aspects,polynucleotide variants are produced by silent substitutions due to thedegeneracy of the genetic code. Polynucleotide variants can be producedfor a variety of reasons, e.g., to optimize codon expression for aparticular host (change codons in the human mRNA to those by a bacterialhost such as E. coli). Vectors and cells comprising the polynucleotidesdescribed herein are also provided.

In some aspects, a DNA sequence encoding a humanized anti-OX40 antibodyor an antigen-binding fragment thereof can be constructed by chemicalsynthesis using an oligonucleotide synthesizer. Such oligonucleotidescan be designed based on the amino acid sequence of the desiredpolypeptide and selecting those codons that are favored in the host cellin which the recombinant polypeptide of interest will be produced.Standard methods can be applied to synthesize an isolated polynucleotidesequence encoding an isolated polypeptide of interest. For example, acomplete amino acid sequence can be used to construct a back-translatedgene. Further, a DNA oligomer containing a nucleotide sequence codingfor the particular isolated polypeptide can be synthesized. For example,several small oligonucleotides coding for portions of the desiredpolypeptide can be synthesized and then ligated. The individualoligonucleotides can contain 5′ or 3′ overhangs for complementaryassembly.

Once assembled (by synthesis, site-directed mutagenesis or anothermethod), the polynucleotide sequences encoding a particular isolatedpolypeptide of interest can be inserted into an expression vector andoperatively linked to an expression control sequence appropriate forexpression of the protein in a desired host. Proper assembly can beconfirmed, e.g., by nucleotide sequencing, restriction mapping, and/orexpression of a biologically active polypeptide in a suitable host. Inorder to obtain high expression levels of a transfected gene in a host,the gene can be operatively linked to or associated with transcriptionaland translational expression control sequences that are functional inthe chosen expression host.

In certain aspects, recombinant expression vectors are used to amplifyand express DNA encoding a humanized anti-OX40 antibody or anantigen-binding fragment thereof. Recombinant expression vectors arereplicable DNA constructs which have synthetic or cDNA-derived DNAfragments encoding a polypeptide chain of a humanized anti-OX40 antibodyor an antigen-binding fragment thereof, operatively linked to suitabletranscriptional or translational regulatory elements derived frommammalian, microbial, viral or insect genes. In one example, atranscriptional unit can comprise an assembly of (1) a genetic elementor elements having a regulatory role in gene expression, for example,transcriptional promoters or enhancers, (2) a structural or codingsequence which is transcribed into mRNA and translated into protein, and(3) appropriate transcription and translation initiation and terminationsequences, as described in detail below. Such regulatory elements caninclude an operator sequence to control transcription. The ability toreplicate in a host, conferred, e.g., by an origin of replication, and aselection gene to facilitate recognition of transformants canadditionally be incorporated. DNA regions are operatively linked whenthey are functionally related to each other. For example, DNA for asignal peptide (secretory leader) is operatively linked to DNA for apolypeptide if it is expressed as a precursor which participates in thesecretion of the polypeptide; a promoter is operatively linked to acoding sequence if it controls the transcription of the sequence; or aribosome binding site is operatively linked to a coding sequence if itis positioned so as to permit translation. Structural elements intendedfor use in yeast expression systems include a leader sequence enablingextracellular secretion of translated protein by a host cell.Alternatively, where a recombinant protein is expressed without a leaderor transport sequence, the protein can include an N-terminal methionine.This methionine can optionally be subsequently cleaved from theexpressed recombinant protein to provide a final product.

The choice of expression control sequence and expression vector willdepend upon the choice of host. A wide variety of expression host/vectorcombinations can be employed. Useful expression vectors for eukaryotichosts, include, for example, vectors comprising expression controlsequences from SV40, bovine papilloma virus, adenovirus andcytomegalovirus. Useful expression vectors for bacterial hosts includeknown bacterial plasmids, such as plasmids from E. coli, including pCR1, pBR322, pMB9 and their derivatives, wider host range plasmids, suchas M13 and filamentous single-stranded DNA phages.

Suitable host cells for expression of a humanized anti-OX40 antibody oran antigen-binding fragment thereof include prokaryotes, yeast, insector higher eukaryotic cells under the control of appropriate promoters.Prokaryotes include gram negative or gram positive organisms, forexample E. coli or bacilli. Higher eukaryotic cells include establishedcell lines of mammalian origin as described below. Cell-free translationsystems could also be employed. Additional information regarding methodsof protein production, including antibody production, can be found,e.g., in U.S. Patent Publication No. 2008/0187954, U.S. Pat. Nos.6,413,746 and 6,660,501, and International Patent Publication No. WO04009823, each of which is hereby incorporated by reference herein inits entirety.

Various mammalian or insect cell culture systems can also be employed toexpress a humanized anti-OX40 antibody or an antigen-binding fragmentthereof. Expression of recombinant proteins in mammalian cells can beperformed because such proteins are generally correctly folded,appropriately modified and completely functional. Examples of suitablemammalian host cell lines include HEK-293 and HEK-293T, the COS-7 linesof monkey kidney cells, described by Gluzman (Cell 23:175, 1981), andother cell lines including, for example, L cells, C127, 3T3, Chinesehamster ovary (CHO), HeLa and BHK cell lines. Mammalian expressionvectors can comprise nontranscribed elements such as an origin ofreplication, a suitable promoter and enhancer linked to the gene to beexpressed, and other 5′ or 3′ flanking nontranscribed sequences, and 5′or 3′ nontranslated sequences, such as ribosome binding sites, apolyadenylation site, splice donor and acceptor sites, andtranscriptional termination sequences. Baculovirus systems forproduction of heterologous proteins in insect cells are reviewed byLuckow and Summers, BioTechnology 6:47 (1988).

A humanized anti-OX40 antibody or an antigen-binding fragment thereofproduced by a transformed host, can be purified according to anysuitable method. Such standard methods include chromatography (e.g., ionexchange, affinity and sizing column chromatography), centrifugation,differential solubility, or by any other standard technique for proteinpurification. Affinity tags such as hexahistidine, maltose bindingdomain, influenza coat sequence and glutathione-S-transferase can beattached to the protein to allow easy purification by passage over anappropriate affinity column. Isolated proteins can also be physicallycharacterized using such techniques as proteolysis, nuclear magneticresonance and x-ray crystallography.

For example, supernatants from systems that secrete recombinant proteininto culture media can be first concentrated using a commerciallyavailable protein concentration filter, for example, an Amicon orMillipore Pellicon ultrafiltration unit. Following the concentrationstep, the concentrate can be applied to a suitable purification matrix.Alternatively, an anion exchange resin can be employed, for example, amatrix or substrate having pendant diethylaminoethyl (DEAE) groups. Thematrices can be acrylamide, agarose, dextran, cellulose or other typescommonly employed in protein purification. Alternatively, a cationexchange step can be employed. Suitable cation exchangers includevarious insoluble matrices comprising sulfopropyl or carboxymethylgroups. Finally, one or more reversed-phase high performance liquidchromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,e.g., silica gel having pendant methyl or other aliphatic groups, can beemployed to further purify a humanized anti-OX40 antibody or anantigen-binding fragment thereof. Some or all of the foregoingpurification steps, in various combinations, can also be employed toprovide a homogeneous recombinant protein.

A humanized anti-OX40 antibody or an antigen-binding fragment thereofproduced in bacterial culture, can be isolated, for example, by initialextraction from cell pellets, followed by one or more concentration,salting-out, aqueous ion exchange or size exclusion chromatographysteps. High performance liquid chromatography (HPLC) can be employed forfinal purification steps. Microbial cells employed in expression of arecombinant protein can be disrupted by any convenient method, includingfreeze-thaw cycling, sonication, mechanical disruption, or use of celllysing agents.

Methods known in the art for purifying antibodies and other proteinsalso include, for example, those described in U.S. Patent PublicationNos. 2008/0312425, 2008/0177048, and 2009/0187005, each of which ishereby incorporated by reference herein in its entirety.

Pharmaceutical Compositions and Administration Methods

Methods of preparing and administering a humanized anti-OX40 antibody oran antigen-binding fragment thereof as provided herein, to a subject inneed thereof, e.g., to enhance an immune response in a cancer patient,e.g., to inhibit or reduce tumor growth, are well known to or can bereadily determined by those skilled in the art. The route ofadministration of a humanized anti-OX40 antibody or an antigen-bindingfragment thereof can be, for example, oral, parenteral, by inhalation ortopical. The term parenteral as used herein includes, e.g., intravenous,intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal, orvaginal administration. While all these forms of administration areclearly contemplated as suitable forms, another example of a form foradministration would be a solution for injection, in particular forintravenous or intraarterial injection or drip. Usually, a suitablepharmaceutical composition can comprise, without limitation, a buffer(e.g. acetate, phosphate or citrate buffer), a surfactant (e.g.polysorbate), a stabilizer agent (e.g. human albumin), etc. In othermethods compatible with the teachings herein, a humanized anti-OX40antibody or an antigen-binding fragment thereof as provided herein canbe delivered directly to the site of the adverse cellular populationthereby increasing the exposure of the diseased tissue to thetherapeutic agent.

Certain pharmaceutical compositions provided herein can be orallyadministered in an acceptable dosage form including, e.g., capsules,tablets, aqueous suspensions or solutions. Certain pharmaceuticalcompositions also can be administered by nasal aerosol or inhalation.Such compositions can be prepared as solutions in saline, employingbenzyl alcohol or other suitable preservatives, absorption promoters toenhance bioavailability, and/or other conventional solubilizing ordispersing agents.

The amount of a humanized anti-OX40 antibody or an antigen-bindingfragment thereof that can be combined with carrier materials to producea single dosage form will vary depending upon the subject treated andthe particular mode of administration. The composition can beadministered as a single dose, multiple doses or over an establishedperiod of time in an infusion. Dosage regimens also can be adjusted toprovide the optimum desired response (e.g., a therapeutic orprophylactic response).

By “therapeutically effective dose or amount” or “effective amount” isintended an amount of a humanized anti-OX40 antibody or anantigen-binding fragment thereof, that when administered brings about apositive therapeutic response with respect to treatment of a patientwith a disease or condition to be treated.

Kits

This disclosure further provides kits that comprise a humanizedanti-OX40 antibody or an antigen-binding fragment thereof as describedherein and that can be used to perform the methods described herein. Incertain embodiments, a kit comprises at least one purified humanizedanti-OX40 antibody or an antigen-binding fragment thereof, in one ormore containers. One skilled in the art will readily recognize that thedisclosed humanized anti-OX40 antibody can be readily incorporated intoone of the established kit formats that are well known in the art.

Immunoassays

A humanized anti-OX40 antibody or an antigen-binding fragment thereofcan be assayed for specific and/or selective binding by any method knownin the art. The immunoassays that can be used include but are notlimited to competitive and non-competitive assay systems usingtechniques such as Western blots, radioimmunoassays, ELISA (enzymelinked immunosorbent assay), fluorescent focus assay (FFA), “sandwich”immunoassays, immunoprecipitation assays, precipitin reactions, geldiffusion precipitin reactions, immunodiffusion assays, agglutinationassays, complement-fixation assays, immunoradiometric assays,fluorescent immunoassays, to name but a few. Such assays are routine andwell known in the art (see, e.g., Ausubel et al., eds, (1994) CurrentProtocols in Molecular Biology (John Wiley & Sons, Inc., NY) Vol. 1,which is incorporated by reference herein in its entirety).

Methods and reagents suitable for determination of bindingcharacteristics of a humanized anti-OX40 antibody as provided herein areknown in the art and/or are commercially available. Equipment andsoftware designed for such kinetic analyses are commercially available(e.g., BIAcore®, BIAevaluation® software, GE Healthcare; KINEXA®Software, Sapidyne Instruments).

Methods of Immune Enhancement and Treatment

The enhancement of an antigen-specific immune response in a subject(e.g., a mammalian subject, such as a human subject) by engaging OX40 onactivated T cells, e.g., activated CD4⁺ T cells and/or activated CD8⁺ Tcells, during or after antigen activation can be accomplished using awide variety of methods. The method of choice will primarily depend uponthe type of antigen against which it is desired to enhance the immuneresponse, and various methods available are discussed below. Whatevermethod is selected, a humanized anti-OX40 antibody or an antigen-bindingfragment thereof can be administered to a subject, e.g., a human patientsuch that it is presented to T cells of the subject during or shortlyafter priming of the T cells by antigen.

In certain aspects, the disclosure provides a method to promote survivalor proliferation of activated T cells, e.g., activated CD4⁺ T cellsand/or activated CD8⁺ T cells, comprising contacting the activated Tcells, e.g., activated CD4⁺ T cells and/or activated CD8⁺ T cells, witha humanized anti-OX40 antibody or an antigen-binding fragment thereof,under conditions where the humanized anti-OX40 antibody can specificallybind to OX40 on the surface of the T cells, e.g., activated CD4⁺ T cellsand/or activated CD8⁺ T cells. In certain aspects the contacting is invitro. In certain aspects the contacting is in vivo, e.g. viaadministration of an effective dose of the humanized anti-OX40 antibodyto a subject in need of treatment. In certain aspects the contacting canoccur at the same time as T cell activation, e.g., antigen activation,in certain aspects the contacting can occur after T cell activation.

In further aspects, the disclosure provides a method of inducingcytokine release from activated T cells, e.g., activated CD4⁺ T cellsand/or activated CD8⁺ T cells, comprising contacting the activated Tcells, e.g., activated CD4⁺ T cells and/or activated CD8⁺ T cells, witha humanized anti-OX40 antibody or an antigen-binding fragment thereof asprovided herein, wherein the humanized anti-OX40 antibody canspecifically bind to OX40 on the surface of the activated T cells, e.g.,activated CD4⁺ T cells and/or activated CD8⁺ T cells. In certain aspectsthe contacting is in vitro. In certain aspects the contacting is invivo, e.g. via administration of an effective dose of the humanizedanti-OX40 antibody to a subject in need of treatment. In certain aspectsthe contacting can occur at the same time as T cell activation, e.g.,antigen activation, in certain aspects the contacting can occur after Tcell activation. In certain aspects the cytokine can be IFNγ, TNFα,IL-5, IL-10, IL-2, IL-4, IL-13, IL-8, IL-12 p70, IL-1β, or anycombination thereof. In certain aspects the cytokine is IFNγ, TNFα,IL-5, IL-10, IL-13, or any combination thereof.

In certain aspects, the activated T cells, e.g., activated CD4⁺ T cellsand/or activated CD8⁺ T cells are human T cells, cynomolgus monkey Tcells, rhesus monkey T cells, or a combination thereof.

The disclosure further provides a method of promoting T cell activation,comprising contacting T cells with a humanized anti-OX40 antibody asprovided herein, wherein the humanized anti-OX40 antibody canspecifically bind to OX40 on the surface of the T cells. In certainaspects the contacting occurs in the presence of antigen, e.g., a tumorantigen. In certain aspects the method further comprises interaction ofan Fc domain of the humanized anti-OX40 antibody with a cell expressingFcγR, e.g., a B cell, monocyte, macrophage, myeloid or plasmacytoiddendritic cell, follicular dendritic cell, Langerhans cell, endothelialcell, NK cell, activated T cell, neutrophil, eosinophil, platelet, mastcell, a CD45⁺ cell from a primary human tumor or tumor-draining ornon-draining lymph node, a CD45⁺ cell from other secondary or tertiarylymphoid structures, or a combination thereof. In certain aspects, the Tcell activation can be measured through stimulation of the NFκB signaltransduction pathway. In certain aspects the contacting is in vitro. Incertain aspects the contacting is in vivo, e.g. via administration of aneffective dose of the humanized anti-OX40 antibody to a subject in needof treatment.

The disclosure further provides a method of treating cancer in asubject, comprising administering to a subject in need of treatment aneffective amount of a humanized anti-OX40 antibody or an antigen-bindingfragment thereof, or a composition or formulation comprising thehumanized anti-OX40 antibody. In certain aspects, the cancer is a solidtumor. According to this method, administration of humanized anti-OX40antibody or composition can inhibit tumor growth; can promote tumorreduction, or both. In certain aspects, the tumor growth inhibition isachieved in the presence of T cells.

The terms “cancer”, “tumor”, “cancerous”, and “malignant” refer to ordescribe the physiological condition in mammals that is typicallycharacterized by unregulated cell growth. Examples of cancers includebut are not limited to, carcinoma including adenocarcinomas, lymphomas,blastomas, melanomas, sarcomas, and leukemias. More particular examplesof such cancers include squamous cell cancer, small-cell lung cancer,non-small cell lung cancer, gastrointestinal cancer, Hodgkin's andnon-Hodgkin's lymphoma, pancreatic cancer, glioblastoma, glioma,cervical cancer, ovarian cancer, liver cancer such as hepatic carcinomaand hepatoma, bladder cancer, breast cancer (including hormonallymediated breast cancer, see, e.g., Innes et al. (2006) Br. J. Cancer94:1057-1065), colon cancer, colorectal cancer, endometrial carcinoma,myeloma (such as multiple myeloma), salivary gland carcinoma, kidneycancer such as renal cell carcinoma and Wilms' tumors, basal cellcarcinoma, melanoma, prostate cancer, vulval cancer, thyroid cancer,testicular cancer, esophageal cancer, various types of head and neckcancer including, but not limited to, squamous cell cancers, and cancersof mucinous origins, such as, mucinous ovarian cancer,cholangiocarcinoma (liver) and renal papillary carcinoma.

This disclosure further provides a method of preventing or treating acancer in a subject in need thereof, comprising administering to thesubject an effective amount of a humanized anti-OX40 antibody or anantigen-binding fragment thereof, a composition or formulationcomprising the humanized anti-OX40 antibody, or a polynucleotide, avector, or a host cell as described herein.

Effective doses of compositions for treatment of cancer vary dependingupon many different factors, including means of administration, targetsite, physiological state of the patient, whether the patient is humanor an animal, other medications administered, and whether treatment isprophylactic or therapeutic. Usually, the patient is a human butnon-human mammals including transgenic mammals can also be treated.Treatment dosages can be titrated using routine methods known to thoseof skill in the art to optimize safety and efficacy.

The compositions of the disclosure can be administered by any suitablemethod, e.g., parenterally, intraventricularly, orally, by inhalationspray, topically, rectally, nasally, buccally, vaginally or via animplanted reservoir. The term “parenteral” as used herein includessubcutaneous, intravenous, intramuscular, intra-articular,intra-synovial, intrasternal, intrathecal, intrahepatic, intralesionaland intracranial injection or infusion techniques.

The disclosure further provides a method of enhancing an immune responsein a subject comprising administering to a subject in need thereof atherapeutically effective amount of a humanized anti-OX40 antibody or anantigen-binding fragment thereof, or a composition or formulationcomprising the humanized anti-OX40 antibody.

The subject to be treated can be any animal, e.g., mammal, in need oftreatment, in certain aspects, subject is a human subject.

In its simplest form, a preparation to be administered to a subject is ahumanized anti-OX40 antibody or an antigen-binding fragment thereof,administered in conventional dosage form, which can be combined with apharmaceutical excipient, carrier or diluent as described elsewhereherein.

A humanized anti-OX40 antibody or an antigen-binding fragment thereofcan be administered by any suitable method as described elsewhereherein, e.g., via IV infusion. In certain aspects, a humanized anti-OX40antibody or an antigen-binding fragment thereof can be introduced into atumor, or in the vicinity of a tumor cell.

All types of tumors are potentially amenable to treatment by thisapproach including, without limitation, carcinoma of the breast, lung,pancreas, ovary, kidney, colon and bladder, as well as melanomas,sarcomas and lymphomas.

Engagement of the OX40 receptor on activated T cells, e.g., activatedCD4⁺ T cells and/or activated CD8⁺ T cells during, or shortly after,priming by an antigen results in an increased response of the activatedT cells, e.g., activated CD4⁺ T cells and/or activated CD8⁺ T cells tothe antigen. In the context of the present disclosure, the term“engagement” refers to binding to and stimulation of at least oneactivity mediated by the OX40 receptor. For example, engagement of theOX40 receptor on antigen specific activated T cells, e.g., activatedCD4⁺ T cells and/or activated CD8⁺ T cells, results in increased T cellproliferation as compared to the response to antigen alone, andincreased cytokine production. The elevated response to the antigen canbe maintained for a period of time substantially longer than in theabsence of OX40 receptor engagement. Thus, stimulation via the OX40receptor enhances the antigen specific immune response by boosting Tcell recognition of antigens, e.g., tumor antigens.

OX40 agonists can enhance antigen specific immune responses in asubject, such as a human subject, when administered to the subjectduring or shortly after priming of T cells by an antigen. OX40 agonistsinclude OX40 ligand (“OX40L”), such as soluble OX40L fusion proteins andanti-OX40 antibodies or fragments thereof. A specific example is ahumanized antibody that specifically binds to OX40, thereby triggeringsignaling. A collection of humanized anti-OX40 monoclonal antibodies areprovided by this disclosure. Also described are nucleic acids includingpolynucleotide sequences that encode such antibodies. This disclosurealso provides methods for enhancing an antigen specific immune responsein a subject using humanized anti-OX40 monoclonal antibodies.

OX40 Epitopes

The portion of a target molecule, e.g., an OX40 polypeptide, whichspecifically interacts with the antigen binding domain of an antibody isan “epitope,” or an “antigenic determinant.” A target molecule, e.g., apolypeptide, can be a single epitope, but typically includes at leasttwo epitopes, and can include any number of epitopes, depending on thesize, conformation, and type of target molecule.

The minimum size of an epitope that can be bound by an antibody on atarget polypeptide is thought to be about four to five amino acids.Peptide or polypeptide epitopes can contain at least seven, at leastnine, at least ten, or at least about 15 or more amino acids. Since anantibody can recognize a polypeptide antigen in its tertiary form, theamino acids comprising an epitope need not be contiguous. An epitope ofOX40, e.g., human OX40 as provided herein can include at least 4, atleast 5, at least 6, at least 7, at least 8, at least 9, at least 10, atleast 15, at least 20, at least 25, or between about 10 to about 30contiguous or non-contiguous amino acids of OX40, e.g., human OX40. Incertain aspects, an epitope of OX40, e.g., human OX40 as provided hereinconsists of a peptide of 100 or fewer amino acids, 75 or fewer aminoacids, 50 or fewer amino acids, 40 or fewer amino acids, 35 or feweramino acids, 30 or fewer amino acids, 25 or fewer amino acids, 20 orfewer amino acids, or 15 or fewer amino acids, and can include at least4, at least 5, at least 6, at least 7, at least 8, at least 9, at least10, at least 15, at least 20, at least 25, or between about 10 to about30 contiguous or non-contiguous amino acids of OX40, e.g., human OX40.On the other hand, an epitope of OX40, e.g., human OX40 as providedherein can include no more than 4, no more than 5, no more than 6, nomore than 7, no more than 8, no more than 9, no more than 10, no morethan 15, no more than 20, no more than 25, or can consist of betweenabout 10 to about 30 contiguous or non-contiguous amino acids of OX40,e.g., human OX40.

In certain aspects, an anti-OX40 antibody or fragment thereof asprovided herein binds to an epitope of OX40, e.g., human OX40, rhesusmonkey OX40, or cynomolgus monkey OX40 that falls within the thirdcysteine rich domain (CRD3) of OX40, e.g., within amino acids 108 to 146of human OX40 (SEQ ID NO: 91), or a peptide at least 17%, at least 75%,at least 80%, at least 85%, and least 90%, at least 95%, or at least100% identical to amino acids 108 to 146 of SEQ ID NO: 91. By “fallswithin” the CRD3 of OX40, means that the means that the epitope caninclude 4 or more, 5 or more, 6 or more 7 or more 8 or more 9 or more,10 or more, 15 or more, or 30 or more contiguous or non-contiguous aminoacids amino acids of the region of OX40 consisting of the CRD3 region,e.g., amino acids 108 to 146 of SEQ ID NO: 91, or a peptide at least70%, at least 75%, at least 80%, at least 85%, and least 90%, at least95%, or at least 100% identical to amino acids 108 to 146 of SEQ ID NO:91.

In certain aspects the OX40 CRD3 peptide that binds the antibodyprovided herein retains a leucine at the position corresponding to aminoacid 116 of SEQ ID NOL 91, and an alanine at the position correspondingto amino acid 126 of SEQ ID NO: 91. For example, certain anti-OX40antibodies or fragments thereof as provided herein bind to human OX40but do not bind to mouse or rat OX40. The CRD3 region of mouse OX40stretches from about amino acid 104 to about amino acid 144 of SEQ IDNO: 92. Amino acid Q113 of mouse OX40, SEQ ID NO: 92, corresponds toamino acid L116 of human OX40, SEQ ID NO: 91, and amino acid V124 ofmouse OX40, SEQ ID NO: 92, corresponds to amino acid A126 of human OX40,SEQ ID NO: 91. As shown in Example 10, an OX40 antibody as providedherein, e.g., OX40mAb24, can bind to a variant of mouse OX40 comprisingSEQ ID NO: 92 except for a Q113L mutation and a V124A.

In certain aspects an isolated peptide is provided, the peptideconsisting of or comprising an epitope that specifically binds an OX40antibody as provided herein, e.g., OX40mAb24. In certain aspects, thepeptide consists of 100 or fewer amino acids, 75 or fewer amino acids,50 or fewer amino acids, 40 or fewer amino acids, 35 or fewer aminoacids, 30 or fewer amino acids, 25 or fewer amino acids, 20 or feweramino acids, or 15 or fewer amino acids, and includes at least 4, atleast 5, at least 6, at least 7, at least 8, at least 9, at least 10, atleast 15, at least 20, at least 25, or between about 10 to about 30contiguous or non-contiguous amino acids of the CRD3 of OX40, e.g., anOX40 region at least 70%, at least 75%, at least 80%, at least 85%, andleast 90%, at least 95%, or at least 100% identical to amino acids 108to 146 of SEQ ID NO: 91. In certain aspects, the peptide retains aleucine at the position corresponding to amino acid 116 of SEQ ID NO:91, and an alanine at the position corresponding to amino acid 126 ofSEQ ID NO: 91.

Such an isolated peptide can be used, e.g., for screening libraries forbinding molecules that specifically bind to OX40, or as an immunogen toraise anti-OX40 antibodies in a subject animal.

This disclosure employs, unless otherwise indicated, conventionaltechniques of cell biology, cell culture, molecular biology, transgenicbiology, microbiology, recombinant DNA, and immunology, which are withinthe skill of the art. Such techniques are explained fully in theliterature. See, for example, Sambrook et al., ed. (1989) MolecularCloning A Laboratory Manual (2nd ed.; Cold Spring Harbor LaboratoryPress); Sambrook et al., ed. (1992) Molecular Cloning: A LaboratoryManual, (Cold Springs Harbor Laboratory, NY); D. N. Glover ed., (1985)DNA Cloning, Volumes I and II; Gait, ed. (1984) OligonucleotideSynthesis; Mullis et al. U.S. Pat. No. 4,683,195; Hames and Higgins,eds. (1984) Nucleic Acid Hybridization; Hames and Higgins, eds. (1984)Transcription And Translation; Freshney (1987) Culture Of Animal Cells(Alan R. Liss, Inc.); Immobilized Cells And Enzymes (IRL Press) (1986);Perbal (1984) A Practical Guide To Molecular Cloning; the treatise,Methods In Enzymology (Academic Press, Inc., N.Y.); Miller and Caloseds. (1987) Gene Transfer Vectors For Mammalian Cells, (Cold SpringHarbor Laboratory); Wu et al., eds., Methods In Enzymology, Vols. 154and 155; Mayer and Walker, eds. (1987) Immunochemical Methods In CellAnd Molecular Biology (Academic Press, London); Weir and Blackwell,eds., (1986) Handbook Of Experimental Immunology, Volumes I-IV;Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., (1986); and in Ausubel et al. (1989) CurrentProtocols in Molecular Biology (John Wiley and Sons, Baltimore, Md.).

General principles of antibody engineering are set forth in Borrebaeck,ed. (1995) Antibody Engineering (2nd ed.; Oxford Univ. Press). Generalprinciples of protein engineering are set forth in Rickwood et al., eds.(1995) Protein Engineering, A Practical Approach (IRL Press at OxfordUniv. Press, Oxford, Eng.). General principles of antibodies andantibody-hapten binding are set forth in: Nisonoff (1984) MolecularImmunology (2nd ed.; Sinauer Associates, Sunderland, Mass.); and Steward(1984) Antibodies, Their Structure and Function (Chapman and Hall, NewYork, N.Y.). Additionally, standard methods in immunology known in theart and not specifically described are generally followed as in CurrentProtocols in Immunology, John Wiley & Sons, New York; Stites et al.,eds. (1994) Basic and Clinical Immunology (8th ed; Appleton & Lange,Norwalk, Conn.) and Mishell and Shiigi (eds) (1980) Selected Methods inCellular Immunology (W.H. Freeman and Co., NY).

Standard reference works setting forth general principles of immunologyinclude Current Protocols in Immunology, John Wiley & Sons, New York;Klein (1982) J., Immunology: The Science of Self-Nonself Discrimination(John Wiley & Sons, NY); Kennett et al., eds. (1980) MonoclonalAntibodies, Hybridoma: A New Dimension in Biological Analyses (PlenumPress, NY); Campbell (1984) “Monoclonal Antibody Technology” inLaboratory Techniques in Biochemistry and Molecular Biology, ed. Burdenet al., (Elsevier, Amsterdam); Goldsby et al., eds. (2000) KubyImmunology (4th ed.; H. Freemand & Co.); Roitt et al. (2001) Immunology(6th ed.; London: Mosby); Abbas et al. (2005) Cellular and MolecularImmunology (5th ed.; Elsevier Health Sciences Division); Kontermann andDubel (2001) Antibody Engineering (Springer Verlag); Sambrook andRussell (2001) Molecular Cloning: A Laboratory Manual (Cold SpringHarbor Press); Lewin (2003) Genes VIII (Prentice Hall 2003); Harlow andLane (1988) Antibodies: A Laboratory Manual (Cold Spring Harbor Press);Dieffenbach and Dveksler (2003) PCR Primer (Cold Spring Harbor Press).

All of the references cited above, as well as all references citedherein, are incorporated herein by reference in their entireties.

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLES

Abbreviations and definitions of terms are listed in Table 2.

TABLE 2 List of Abbreviations and Definitions of Terms Abbreviation orTerm Definition 1A7 Isotype control mouse IgG1 kappa antibody 9B12 Mouseanti-human OX40 IgG1κ monoclonal antibody A375 human melanoma cell lineAa amino acid ADCC Antibody-dependent cellular cytotoxicity ADPEAntibody Development and Protein Engineering ANOVA Analysis of varianceAsp aspartic acid BSA Bovine serum albumin BLASTP protein sequencehomology Basic Local Alignment Search Tool C Final tumor volumes fromthe control group CD cluster of differentiation CDC Complement-dependentcytotoxicity CD4⁺, OX40⁺ CD4 positive, OX40 positive CDRcomplementarity-determining regions CFA Complete Freund's adjuvant CFSECarboxyfluorescein succinimidyl ester CI Confidence Interval ClqComplement component Clq CR Complete response CRD Cysteine rich domainCyno cynomolgus DM-L density media-lymphocyte E:T effector-to-target(ratio) EC effective concentration ECf effective concentration resultingin f % of maximal effect EC₂₀ effective concentration resulting in 20%of maximal effect EC₅₀ half maximal effective concentration EC₉₀effective concentration resulting in 90% of maximal effect F fraction ofmaximal FACS fluorescence activated cell sorting FBS fetal bovine serumFc Fragment, crystallizable Fcer1g−/− Genetically engineered mousestrain; lacks expression of activating Fc gamma receptors (Fc gamma I,III, and IV) Fcgr2b−/− Genetically engineered mouse strain; lacksexpression of inhibitory Fc gamma IIb receptor FCS Flow cytometrystandard Fcγ fragment, crystallizable gamma FMO Fluorescence minus-oneFP Fusion protein G Force of gravity H hour H⁺L heavy plus light chainsHEK293 Human embryonic kidney cell line Hr hour HSC Hematopoietic stemcell Hu Human ICOS Inducible T-cell co-stimulator IgG ImmunoglobulinIgG1 Immunoglobulin G1 IL-2 interleukin 2 IP intraperitoneal IUInternational Units K_(d) equilibrium binding dissociation constant KIknock-in KLH Keyhole limpet hemocyanin KO knock-out K_(p) equilibriumdissociation constant Leu Leucine M Mouse mAb monoclonal antibody mLmilliliter OX40mAb24 Humanized anti-human OX40 IgG1κ monoclonal antibodyMFI mean fluorescence intensity mOX40L FP Mouse OX40 ligand mouse IgG1fusion protein mOX40L FP Mouse OX40 ligand mouse IgG1 fusion protein(Y182A) engineered to have reduced binding to OX40 NFκB Nuclear factorkappa-light-chain-enhancer of activated B cells NIP228 Mouse IgG1 kappamonoclonal antibody against 4-hydroxy-3-iodo-5-nitrophenylacetic acid NKnatural killer NOD/SCID non-obese diabetic/severe combinedimmunodeficient NSG Mice with genetic backgroundNod.Cg-Prkd^(cscid)112rg^(tm/Wy)/SzJ OX40L OX40 ligand OX40L FP humanOX40 ligand IgG4P fusion protein engineered IgG4P Y180A to have reducedbinding to OX40 OX40mAb24 Humanized anti-human OX40 IgG1 κ monoclonalantibody OX86 Rat anti-mouse OX40 IgG1κ monoclonal antibody PBMCperipheral blood mononuclear cells PBS Phosphate buffered saline PHA-LPhytohemagglutinin-Leucoagglutinin PI Propidium iodide PK/PDPharmacokinetic/pharmacodynamics RBC red blood cell RBCL Red blood celllysis RBD receptor binding domain Rh recombinant human RLU Relativelight units RPMI Roswell Park Memorial Institute 1640 medium ROA Routeof administration SC subcutaneous SD Standard Deviation SEM StandardError of the Mean Ser serine T Final tumor volumes from the test groupTcm Central Memory T cell TCR T cell receptor Teff Effector T cell TemEffector memory T cell TGI tumor growth inhibition TIL tumorinfiltrating leukocyte TM transmembrane domain TNFR tumor necrosisfactor receptor TNFRSF tumor necrosis factor receptor superfamily TRAF2tumor necrosis factor receptor-associated factor 2 Treg regulatory Tcells μL microliter μg Microgram V volume

Example 1 Humanization of Anti-human OX40 Murine MAb 9B12

Murine mAb 9B12 was humanized by grafting its CDRs onto selected humangermline frameworks. The sequences of the heavy chain variable region(VH) and the light chain variable region (VL) of murine mAb 9B12 werecompared with the human antibody germline sequences available in thepublic NCBI databases. Human acceptor frameworks (FR) were identifiedbased on the highest sequence homology. When selecting an optimalacceptor framework several criteria were considered such as matchingresidues that could impact binding (residues in the Vernier zone,canonical class residues, and VH/VL interface residues), and thepotential for immunogenicity (low germline frequency). An optimal hybridhuman acceptor FR sequence was designed for VH and VL independently byselecting the most homologous human immunoglobulin germline segment foreach individual framework region. Three different germline acceptorsequences were combined to form the humanized VH framework backbone,while two different germline acceptor sequences were selected for VL.The fully human germline acceptor template chosen for the VH chain(9B12VH-hu) was a combination of IGVH4-34*09 (FR 1), VH4-39 (FR 2),VH6-1(FR 3) and JH4 (FR 4). VL (9B12VL-hu1) was a combination of 018 (FR1), 018 (FR 2), L23 (FR 3) and JK1 (FR 4). The framework homologybetween the murine sequence and human template acceptor sequence wasapproximately 72% for VH and 77% for VL.

Murine framework residues which could influence or maintain thefunctional conformation of the parent CDR and did not match the humangermline sequence were identified and selectively reintroduced into thehuman acceptor FR to best preserve the 9B12 binding affinity andfunctionality. In this case, mouse FR residues 27D, 39K, 47Y, 48M, 71R,78Y and 91F in the VH chain and 44V and 68R in the VL chain were mutatedback in the human template. CDR residues as defined by Kabat were fusedinto the designed acceptor frameworks for both VH and VL for generatingthe humanized antibody. Two humanized VH genes (9B12VH-hu and9B12VH-hu39K71R) and two VL genes (9B12VL-hu1 and 9B12VL-hu2) weresynthesized by GeneART (Thermo Fisher Scientific, Waltham, Mass.), thencloned into the in-house pOE IgG1 expression vector.

A panel of humanized variants has been generated and characterized in Tcell-based binding and proliferation assays. The humanized variants,containing several framework amino acids reverted to mouse FR residuesin the heavy chain variable region, bound to OX40 on activated CD4⁺ Tcells with comparable affinities and similar potencies (T cellproliferation) to 9B12. The variable light chain paired with allhumanized VH variants encodes for fully humanized frameworks. To reducethe risk of immunogenicity, the number of mouse residues in the VH humanFR was further reduced to 3 or 4 by replacing non-influential mouseresidues with the corresponding human residues. To remove potentialsequence liabilities, the NG deamidation site in VH-CDR2, the RYDintegrin binding site and the DG isomerization site in VH-CDR3 wereproactively removed either independently or in combination. To avoidADCC mediated by human IgG1 Fc effector function, IgG4P and IgG1TM Fcvariants were made for the humanized lead mAbs. The resultant IgG4P andIgG1TM variants exhibited the same binding activity to OX40 as the IgG1variants, but significantly reduced ADCC activity which is most similarto the murine mAb 9B12. In summary, the humanized variants exhibitedcell-binding affinities and in vitro potencies comparable to the parentmouse mAb 9B12. The amino acid differences among the humanized VHvariant, all of which are paired with the humanized VL, are summarizedin FIG. 1.

Reverse chimera was engineered for in vivo characterization in rhesusmonkeys. Briefly, the VH and VL of humanized mAb24 were grafted onto theconstant heavy and constant light chains of murine 9B12. In vitrocharacterization demonstrated the Fab portions of the reverse chimerabound to human OX40 comparable to mAb24.

Example 2 Binding Affinity and Receptor Occupancy of OX40mAb24 to NativeOX40 Expressed on the Surface of Activated Human, Non-human Primate, Ratand Mouse T Cells

2.1 Materials

Materials used in this example are listed in Table 2-1.

TABLE 2-1 Materials Item Source AlexaFluor ® A488 goat anti-human IgG(H + L) Life Technologies, Carlsbad, CA AlexaFluor ® A488 goatanti-mouse IgG (H + L) Life Technologies, Carlsbad, CA AlexaFluor ® A488goat anti-rat IgG (H + L) Life Technologies, Carlsbad, CAAntibiotic/antimycotic solution, 100X Life Technologies, Carlsbad, CAAnti-rat CD134 (OX40 clone) antibody Biolegend, San Jose, CA Anti-ratCD3 antibody BD Biosciences, San Jose, CA Anti-mouse CD134 (OX86 clone)antibody MedImmune, Gaithersburg, MD Anti-rat IgG1, κ isotype controlmAb clone Biolegend, San Jose, CA RTK2071 Balb/C mouse Harlan,Indianapolis, IN Beta mercaptoethanol (BME) Life Technologies, Carlsbad,CA Concanavalin A Sigma, St. Louis, MO Ethylenediaminetetraacetic acid(EDTA) Life Technologies, Carlsbad, CA Heat inactivated newborn calfserum (FBS) Life Technologies, Carlsbad, CA Hamster anti-mouse CD3antibody BD Biosciences, San Jose, CA Hamster anti-mouse CD28 antibodyBD Biosciences, San Jose, CA IL-2, recombinant human Preprotech, RockyHill, NJ Lymphocyte separation medium (LSM) MP Biomedicals, Santa Ana,CA Magcellect rat CD4 T cell isolation kit R&D Systems, Minneapolis, MNMiltenyi MACS buffer Miltenyi San, Diego, CA Mouse CD4 T cell isolationkit Miltenyi San, Diego, CA Mouse IgG1, κ isotype control mAb cloneMOPC-21 Biolegend, San Jose, CA Non-human primate CD4 T cell isolationkit Miltenyi San, Diego, CA Non-TC treated round-bottom 96 well platesVWR, Radnor, PA Percoll Sigma, St. Louis, MOPhytohemagglutinin-Leucoagglutinin (PHA-L) Roche Applied Science,Indianapolis, IN Phosphate buffered saline, pH 7.2 (PBS) LifeTechnologies, Carlsbad, CA RosetteSep CD4 T cell enrichment kit StemCell Technologies, Vancouver, BC RPMI-1640 Life Technologies, Carlsbad,CA Sprague Dawley rat Harlan, Indianapolis, IN Whole blood, sodiumheparin anti-coagulated MedImmune Blood Donor Program, Gaithersburg, MD

In this example, cell-based equilibrium binding assays were performed tomeasure the apparent affinity of OX40mAb24 binding to OX40 expressed onthe cell surface of human, non-human primate, rat and mouse T cells.Additionally, the equilibrium binding assays were utilized to determinethe concentrations of OX40mAb24 that achieve 20%, 50%, or 90% human OX40receptor occupancy on activated human CD4⁺ T cells.

OX40mAb24 concentrations required to achieve 20%, 50% or 90% receptoroccupancy were also determined for binding of 9B12, the murineanti-human OX40 monoclonal antibody from which OX40mAb24 was derived, tohuman and non-human primate OX40 expressed on the surface of CD4⁺ Tcells for a comparison of OX40mAb24 and 9B12 binding.

2.2 Assays

2.2.1 Binding of OX40mAb24 to Primary Human CD4⁺ T Cells andOX40-expressing Jurkat T Cells

The apparent equilibrium binding consent (K_(d)) for OX40mAb24 bindingto human OX40 and the concentrations required to bind 20%, 50%, or 90%of cell surface human OX40 receptor at equilibrium were calculated frombinding curves of OX40mAb24 to OX40-expressing activated primary humanCD4⁺ T cells or human OX40-overexpressing Jurkat T cells. Similarexperiments were performed at the same time to assess binding of 9B12 tothese cells for comparison with OX40mAb24 values.

Primary human CD4⁺ T cells were first isolated from sodium heparinanti-coagulated whole blood obtained from healthy donors through theMedImmune Blood Donor Program using a RosetteSep CD4⁺ T cell enrichmentkit (Stem Cell Technologies, Vancouver, BC) and a modifiedmanufacturer's protocol.

Primary human CD4⁺ T cells were cultured for 48 h with 2 μg/mL PHA-L and20 IU/mL rhIL-2 to activate T cells and up-regulate OX40. Activated Tcells, which were >95% viable, were subsequently used in OX40mAb24binding experiments. All donors represent unique individuals; that is,repeat binding experiments were not performed with CD4⁺ T cells from thesame donor.

Human OX40-overexpressing Jurkat NFκB-luciferase clone 64 cells werecultured in complete RPMI+10% FBS prior to binding experiments, withoutthe need for activation.

OX40mAb24 (10 μg/mL) or 9B12 (10 μg/mL) was diluted over a 17 point2-fold dilution series. OX40mAb24 was added to 100,000 cells (activatedprimary CD4⁺ T cells or human OX40-overexpressing Jurkat NFkB-luciferaseclone 64 cells) per well and incubated for one hour at 4° C. Forbackground binding subtraction, cells were incubated in the presence ofsecondary antibody alone. The control R347 human IgG1 monoclonalantibody and mouse IgG1 isotype clone MOPC-21 were used in experimentswith OX40 over-expressing Jurkat T cells to demonstrate the specificityfor OX40 binding of OX40mAb24 or 9B12, respectively. Followingincubation, cells were washed three times with 200 μL of cold (4° C.)FACS buffer and incubated with 100 μL of FACS buffer (PBS+2%heat-inactivated newborn calf serum) containing 10 μg/mL AlexaFluor® 647labeled goat anti-human IgG secondary antibody (for binding toOX40mAb24) or 10 μg/mL AlexaFluor® 488 labeled goat anti-mouse IgGsecondary antibody (for binding to 9B12) and 5 μg/mL propidium iodide(PI). Following secondary antibody incubation, cells were washed andsuspended in 100 μL FACS buffer for flow cytometry analysis on a BDLSRII flow cytometer as described below.

2.2.2 OX40mAb24 Binding to Mouse CD4⁺ T Cells

OX40mAb24 was investigated for binding to mouse OX40 expressed onactivated primary CD4⁺ T cells. Similar experiments were performed atthe same time to assess binding of 9B12 to these cells in comparisonwith OX40mAb24. Mouse CD4⁺ T cells were isolated from harvested normalBalb/C mouse spleens according to the following protocol:

Spleens were mashed against a 70 μM nylon filter to release splenocytesand the filter rinsed with 1 mL complete medium (RPMI-1640 plus 10% FBS,1% antibiotic/antimycotic solution and 55 μM beta mercaptoethanol[BME]). Splenocytes were pelleted and the supernatant discarded. Thepellet was treated with 5 mL of 1× red blood cell (RBC) lysis buffer andincubated to lyse RBCs. Osmolarity was restored by addition of completemedium at the end of incubation time.

Cells were pelleted, washed in Miltenyi MACS buffer (PBS pH 7.2+0.5%bovine serum albumin (BSA)+2 mM ethylenediaminetetraacetic acid [EDTA])and the supernatant was discarded. The pellet was suspended in cold MACSbuffer and counted with a ViCell counter to determine cell number andviability.

Mouse CD4⁺ T cell isolation was performed with a Miltenyi process kit(San Diego, Calif.) according to manufacturer's instructions, andisolated cells were suspended in complete medium

Mouse CD4⁺ T cells (150,000 per well in 100 μL complete medium) werecultured overnight in 96-well plates coated with 2 μg/mL each hamsteranti-mouse CD3 and hamster anti-mouse CD28 antibodies to activate Tcells and induce OX40 expression. Activated CD4⁺ T cells were removedfrom the incubation plate and 100,000 cells were transferred to eachwell of a non-tissue culture-treated 96-well round-bottom plate forbinding assays and washed once with FACS buffer. Binding was performedwith 10 μg/mL of OX40mAb24, 9B12 and rat anti-mouse OX40, clone OX86(positive control) antibodies each serially diluted 3-fold in FACSbuffer for a 10-point data curve. For negative controls, 10 μg/mL R347human IgG1, MOPC-21 mouse IgG1 or RTK2071 rat IgG1 were diluted 6-foldfor a 3 point data curve. FACS buffer (50 μL) containing OX40mAb24, orantibodies, was added to CD4⁺ T cells in duplicate and incubated.Following primary incubation, cells were washed with 200 μL of 4° C.FACS buffer and incubated with 50 μL of FACS buffer containing 10 μg/mLAlexaFluor® 488 labeled goat anti-human secondary, 10 μg/mL AlexaFluor®488 labeled goat anti-mouse secondary or AlexaFluor® 488 labeled goatanti-rat secondary antibody and 5 μg/mL PI. Following secondary antibodyincubation, cells were washed with 4° C. FACS buffer (200 μL per wash)and suspended in 100 μL FACS buffer for flow cytometry analysis on a BDLSRII flow cytometer as described in Section 2.3.

2.2.3 OX40mAb24 Binding to Rat CD4⁺ T cells

OX40mAb24 was investigated for binding to rat OX40 expressed onactivated primary CD4⁺ T cells. Similar experiments were performed atthe same time with 9B12 to allow comparison with OX40mAb24. Rat CD4⁺cells were isolated from freshly harvested normal Sprague-Dawley ratspleens according to the protocol described above for the isolation ofmouse splenocytes, except that rat CD4⁺ T cell isolation was performedwith an R&D Systems Magellect kit (Minneapolis, Minn.) according tomanufacturer's instructions.

Rat CD4⁺ T cells (1×10⁶ per mL complete medium) were cultured overnightin a T75 cell culture flask with 1 μg/mL concanavalin A (Con A) and 500IU/mL IL-2, to activate T cells and induce OX40 expression, andincubated overnight. Activated CD4⁺ T cells were removed from the flaskand 100,000 cells were transferred to each well of a non-tissue culturetreated 96-well round-bottom plate for binding assays and washed withFACS buffer. Binding was performed with 10 μg/mL OX40mAb24, 9B12 ormouse anti-rat CD134, clone OX40 (positive control) antibody seriallydiluted 3-fold for a 10 point data curve in FACS buffer. For negativecontrols, 10 μg/mL R347 human IgG1 or mouse IgG1 clone MOPC-21 wereserially diluted 6-fold for a 3 point data curve. 100 μL of OX40mAb24control protein, 9B12 or clone OX40 antibody was added to CD4⁺ T cellsin duplicate and incubated. Following primary incubation, cells werewashed with 200 μL of 4° C. FACS buffer per wash and incubated with 100μL of FACS buffer containing 10 μg/mL AlexaFluor® 488 labeled goat andhuman, or AlexaFluor® 488 labeled goat anti-mouse secondary antibody and5 μg/mL PI. Following secondary antibody incubation, cells wereprocessed for flow cytometry as described in Section 2.3.

2.2.4 OX40mAb24 Binding to Cynomolgus Monkey CD4⁺ T Cells

OX40mAb24 was investigated for binding to cynomolgus monkey (cyno) OX40expressed on activated primary CD4⁺ T cells. Similar experiments wereperformed at the same time with 9B12 to allow comparison with OX40mAb24values. Cyno CD4⁺ T cells were isolated from sodium heparinanti-coagulated whole blood obtained from healthy cyno donors (N=2) fromWorld Wide Primates (Miami, Fla.) according to the following protocol:

Whole blood was layered onto 30 mL of 60% Percoll in a 50 mL conicalcentrifuge tube. Blood was centrifuged and peripheral blood mononuclearcells (PBMC) were collected at the interface and washed with cold (4°C.) Miltenyi MACS buffer at 1200 RPM for 10 minutes. Supernatant wasdiscarded and the pellet was treated with 5 mL of 1× RBC lysis bufferand incubated. Complete medium (RPMI with 10% FBS and 1%antibiotics/antimycotics) was added to the pellet to stop the lysisprocess at the end of incubation time.

Cells were pelleted and washed with 20 mL of cold (4° C.) Miltenyi MACSbuffer. The supernatant was discarded and the cell pellet was suspendedin cold (4° C.) MACS buffer and counted with a ViCell counter todetermine cell number and viability.

Cyno CD4⁺ T cell isolation was performed with a Miltenyi non-humanprimate kit according to manufacturer's instructions, then CD4⁺ T cellscounted on a ViCell counter and suspended at 1×10⁶ per mL in completemedium as described above.

Cyno CD4⁺ T cells (1×10⁶ per mL in complete medium) were incubated for48 hours in a T75 cell culture flask with 2 μg/mL PHA-L and 20 IU/mLIL-2, to activate T cells and induce OX40 expression. Activated CD4⁺ Tcells were removed from the flask and 100,000 cells were transferred toeach well of a non-tissue culture treated 96-well round-bottom plate forbinding assays and washed with 200 μL FACS buffer. Binding was performedwith 10 μg/mL OX40mAb24 or 9B12 serially diluted 4-fold in FACS bufferfor an 11-point data curve, or both R347 human IgG1 or mouse IgG1 cloneMOPC-21 (negative controls) serially diluted 6-fold for a 3 point datacurve. OX40mAb24, 9B12 or control protein was added to CD4⁺ T cells andincubated. Following primary incubation, cells were washed with 200 μLof cold (4° C.) FACS buffer per wash and incubated with 100 μL of FACSbuffer containing 10 μg/mL AlexaFluor® 488 labeled goat anti-humansecondary antibody or 10 μg/mL AlexaFluor® 488 labeled goat anti-mousesecondary and 5 μg/mL PI. Following secondary antibody incubation, cellswere processed for flow cytometry as described in Section 2.3.

2.2.5 OX40mAb24 Binding to Rhesus Monkey CD4⁺ T Cells

OX40mAb24 was investigated for binding to rhesus macaque OX40 expressedon activated primary CD4⁺ T cells. Similar experiments were performed atthe same time with 9B12 to allow comparison with OX40mAb24 values.Rhesus CD4⁺ T cells were isolated from sodium heparin anti-coagulatedwhole blood obtained from healthy rhesus donors (N=2) from World WidePrimates (Miami, Fla.) according to the following protocol:

Heparinized rhesus blood (20 mL) was diluted 1:1 with PBS and layeredonto 15 ml of 95% LSM in a 50 ml conical centrifuge tube. Blood wascentrifuged, PBMC were collected at the interface and washed twice withcold Miltenyi MACS buffer at 400×g for 30 minutes. Supernatant wasdiscarded and the pellet was treated with 5 mL of 1× RBC lysis bufferand incubated. Complete medium (RPMI with 10% FBS and 1%antibiotics/antimycotics) was added to the pellet to stop the lysisprocess at the end of incubation time. Cells were pelleted and washedwith 20 mL of cold Miltenyi MACS buffer. Supernatant was discarded andthe pellet was suspended in cold MACS buffer and counted with a ViCellcounter to determine cell number and viability. Rhesus CD4⁺ T cellisolation was performed with a Miltenyi non-human primate kit (SanDiego, Calif.) according to manufacturer's instructions.

Rhesus CD4⁺ T cells (1×10⁶ per mL in complete medium) were cultured for48 hours in a T75 cell culture flask with 5 μg/mL Con-A and 1000 IU/mLIL-2 to activate T cells and induce OX40 expression. Binding to 100,000activated rhesus CD4⁺ T cells was performed with 10 μg/mL OX40mAb24 and9B12 serially diluted 3-fold in FACS buffer for a 10 point(experiment 1) or 12 point data curve (experiment 2) and 10 μg/mL humanand mouse isotype controls diluted 6-fold for 2 data points(experiment 1) or 4 data points (experiment 2). AlexaFluor® 488-labeledgoat anti-human secondary antibody and AlexaFluor® 488 labeled goatanti-mouse secondary binding were as described above for cyno CD4⁺ Tcells, and flow cytometry as described in Section 2.3.

2.3 Flow Cytometry

Flow cytometry in the assays described in Section 2.1 was performedusing an LSRII flow cytometer (Becton-Dickinson, San Jose, Calif.). FlowJo cytometry analysis software (TreeStar, Ashland, Oreg.) was used todetermine OX40mAb24, 9B12 and control protein binding to cells. Wellscontaining OX40-expressing cells (unstained, no PI or secondaryantibody), cells bound to AlexaFluor® 488- or AlexaFluor® 647-labeledsecondary antibody reagent only or cells permeabilized with 0.1% saponinand treated with 10 μg/mL PI were prepared for single-stain compensationcontrols. After fluorescence compensation, live (PI negative) cells weregated and the mean fluorescence intensity (MFI) of secondary antibodywas determined.

2.4 Calculations

2.4.1 Determination of Apparent Equilibrium Dissociation Constant(K_(d))

GraphPad Prism version 5.01 for Windows, GraphPad Software, San DiegoCalif. USA, www.graphpad.com, was used to plot MFI of OX40mAb24 bindingversus protein concentration (M) to create binding curves from whichapparent K_(d) was determined. To determine the apparent K_(d) forOX40mAb24 and 9B12 binding to human, mouse, rat, cyno, or rhesus monkeyOX40, a non-linear regression (curve fit) equation for one site(specific) binding was employed.

2.4.2 Determination of 20%, 50%, and 90% Receptor Occupancy Values

The amount of a monoclonal antibody (mAb) bound to its receptor can beestimated from the following binding relationship:Receptor(A)+mAb(B)

receptor-mAb complex (AB)  Equation 1

The binding dissociation constant (K_(d)) of the respective antibody isrepresented by:

$\begin{matrix}{{K\; d} = \frac{\lbrack A\rbrack\lbrack B\rbrack}{\left\lbrack {A\; B} \right\rbrack}} & {{Equation}\mspace{14mu} 2}\end{matrix}$Where [A] and [B] represent the concentrations of free receptor andantibody, respectively. Finally, the fractional occupancy, fraction (F)of all receptor molecules that are bound to the antibody, can becalculated by:

$\begin{matrix}{F = \frac{\left\lbrack {A\; B} \right\rbrack}{\lbrack A\rbrack + \left\lbrack {A\; B} \right\rbrack}} & {{Equation}\mspace{14mu} 3}\end{matrix}$Formation of Equation 2 and Substitution into Equation 3 Results in:

$\begin{matrix}{F = \frac{{{\lbrack A\rbrack\lbrack B\rbrack}/K}\; d}{\lbrack A\rbrack + \left( {{{\lbrack A\rbrack\lbrack B\rbrack}/K}\; d} \right)}} & {{Equation}\mspace{14mu} 4}\end{matrix}$Simplification of Equation 4 Results in:

$\begin{matrix}{F = \frac{\lbrack B\rbrack}{\lbrack B\rbrack + {K\; d}}} & {{Equation}\mspace{14mu} 5}\end{matrix}$Therefore, at equilibrium condition, the fraction of all receptormolecules that are bound to the antibody can be calculated if theconcentration of free mAb and the dissociation constant K_(d) of therespective antibody are known.Derivation of this formula for the calculation of the concentration ofantibody required for a fractional receptor occupancy expressed as F,leads to the following formula:[B]=F*Kd/(1−F)  Equation 6Where [B] equals the concentration of mAb, in this case OX40mAb24. Thisformula (Equation 6) was used for the calculation of the concentrationsof OX40mAb24 required for 20, 50, and 90% receptor occupancy (F=0.20,0.50 or 0.90) from cell binding experiments from which the K_(d) valuewas calculated using the non-linear regression (curve fit) equation forone site (specific) binding described above.2.5 Statistical Methods

A 2-sided unpaired Student's t test with 95% confidence level andWelch's correction to account for data sets with different standarddeviations was utilized in Graphpad Prism software to determinestatistically significant differences between apparent K_(d) values orapparent receptor occupancy values determined for OX40mAb24 binding toOX40 on activated primary human CD4 T cells versus OX40-expressingJurkat T cells. Descriptive statistics (i.e., mean and standard error ofthe mean) are presented in the summary figures and tables.

2.6 Results

2.6.1 Binding of OX40mAb24 to Primary Human CD4⁺ T Cells

OX40mAb24 bound to activated human CD4⁺ T cells with a mean apparentK_(d) of 312 pM and 20%, 50% and 90% receptor occupancy values of 78.1,312, and 2810 pM, respectively; n=6 binding assays with six independentT cell donors (FIGS. 2A and 2B, and Table 2-2).

In comparison, 9B12 bound to activated human CD4⁺ T cells with a meanK_(d) of 669 pM, and 20%, 50% and 90% receptor occupancy values of 167,669, and 6020 pM, respectively (FIGS. 2C and 2D and Table 2-2). Theratio between 9B12 and OX40mAb24 apparent K_(d) values was therefore 2.1to 1, and reflects a similar binding affinity to human OX40 for themurine and humanized monoclonal antibodies

TABLE 2-2 Apparent Affinity (K_(d)) and Receptor Occupancy Values forBinding of OX40mAb24 or 9B12 to OX40-expressing Activated Primary HumanCD4⁺ T cells Binding N K_(d) (StdErr), EC₂₀ (StdErr), EC₅₀ (StdErr),EC₉₀ (StdErr), Protein donors pM pM pM pM OX40mAb24 6 312 (57.9) 78.1(14.5) 312 (57.9) 2810 (521)  9B12 6 669 (137)   167 (34.3) 669 (137) 6020 (1230) EC₂₀ = effective concentration resulting in 20% of maximaleffect; EC₅₀ = half maximal effective concentration; EC₉₀ = effectiveconcentration resulting in 90% of maximal effect; K_(d) = equilibriumbinding dissociation constant; StdErr = standard error of the mean.2.6.2 Binding of OX40mAb24 to a Jurkat T Cell Line Engineered toOver-Express Human OX40

OX40mAb24 bound to OX40-expressing Jurkat T cells with a mean K_(d) of424 pM and 20%, 50% and 90% receptor occupancy values of 106, 424, and3820 pM, respectively. FIGS. 3A-C and Table 2-3. There were nostatistically significant differences in apparent binding affinity orreceptor occupancy of OX40mAb24 to OX40 expressed by activated primaryhuman CD4⁺ T cells and OX40 over-expressing Jurkat T cells (p=0.59).

In comparison, 9B12 bound these cells with a mean K_(d) of 726 pM and20%, 50% and 90% receptor occupancy values of 182, 726, and 6540 pM,respectively (FIGS. 3D-F and Table 2-3). The ratio between the apparentK_(d) values for 9B12 and OX40mAb24 was therefore 1.7 to 1, similar tothe ratio calculated for binding to OX40 on activated human CD4⁺ T cells

TABLE 2-3 Apparent Affinity (K_(d)) and Receptor Occupancy Values forBinding of OX40mAb24 or 9B12 to Human OX40-overexpressing JurkatNFkB-luciferase Clone 64 cells Binding N K_(d) (StdErr), EC₂₀ (StdErr),EC₅₀ (StdErr), EC₉₀ (StdErr), Protein experiments pM pM pM pM OX40mAb243 424 (173) 106 (43.3) 424 (173) 3820 (1560) 9B12 3 726 (308) 182 (76.9)726 (308) 6540 (2770) EC₂₀ = effective concentration resulting in 20% ofmaximal effect; EC₅₀ = half maximal effective concentration; EC₉₀ =effective concentration resulting in 90% of maximal effect; K_(d) =equilibrium binding dissociation constant; StdErr = standard error ofthe mean.2.6.3 Binding of OX40mAb24 to Mouse or Rat CD4⁺ T Cells

Neither OX40mAb24 nor 9B12 bound to activated mouse or rat CD4⁺ T cells(data not shown). Positive staining of activated mouse and rat CD4⁺ Tcells was observed with commercial anti-mouse and anti-rat OX40antibodies, clones OX86 and OX40, respectively. 9B12 did not bindactivated mouse nor rat CD4⁺ T cells (data not shown).

2.6.4 Binding of OX40mAb24 to Cynomolgus and Rhesus Monkey CD4⁺ T Cells

OX40mAb24 bound to activated cynomolgus cells with a mean K_(d) of 581pM. The cyno K_(d) was 1.9-fold higher than the human K_(d) (Table 2-4).

9B12 bound the activated cyno CD4⁺ T cells with a mean K_(d) of 1088 pM(Table 2-4), which resulted in a ratio between 9B12 and OX40mAb24apparent K_(d) values of 1.9 to 1.

OX40mAb24 bound to activated rhesus monkey CD4⁺ T cells with a meanK_(d) of 369 pM (Table 2-4). The rhesus K_(d) was 1.2-fold higher thanthe human K_(d).

9B12 bound the activated rhesus monkey CD4⁺ T cells with a mean K_(d) of713 pM (Table 2-4), which results in a ratio between 9B12 and OX40mAb24apparent K_(d) values not shown of 2.8 to 1

TABLE 2-4 Apparent Affinity (K_(d)) for Binding of OX40mAb24 or 9B12 toCynomolgus and Rhesus Monkey OX40-expressing Activated Primary CD4⁺ Tcells Cynomolgus Rhesus N K_(d) (StdErr), N K_(d) (StdErr), BindingProtein experiments pM experiments pM OX40mAb24 2 581 (238) 2 369 (236)9B12 2 1088 (37)  2 713 (559) K_(d) = equilibrium binding dissociationconstant; StdErr = standard error of the mean.

Example 3 Binding Specificity of OX40mAb24 for Human OX40

In this example, flow cytometry-based cell binding assays were performedto determine the specificity of OX40mAb24 for human OX40, relative toother human TNFRSF members with related amino acid sequences, whichincluded: NGFR (TNFRSF16), LTBR (TNFRSF3), TNFR2 (TNFRSF1B), GITR(TNFRSF18), CD137 (TNFRSF9), and HVEM (TNFRSF14). In addition, thebinding specificity of OX40mAb24 for recombinant human TNFRSF memberswas tested in an ELISA format, which included those mentioned above aswell as DR6 (TNFRSF21), osteoprotegerin (OPG; TNFRSF11B), RANK(TNFRSF11A), FAS (TNFRSF6), and CD40 (TNFRSF5).

3.1 Materials

Materials used in this study are listed in Table 3-1.

TABLE 3-1 Materials Item Source AlexaFluor ® 647 goat anti-human IgG(H + L) Life Technologies, Carlsbad, CA BioTek ® plate washer BioTek ®,Wincoski, VT CD137 (TNFRSF9) pCMV6-XL5 expression vector OrigeneTechnologies, Inc., Rockville, MD GITR (TNFRSF18) pCMV6-XL5 expressionvector Origene Technologies, Inc., Rockville, MD Goat anti-human IgGkappa light chain HRP conjugate Sigma-Aldrich, St. Louis, MOGoat-antimouse IgG (Fab specific) HRP conjugate Sigma-Aldrich, St.Louis, MO HVEN (TNFRSF14) pCMV6-XL4 expression vector OrigeneTechnologies, Inc., Rockville, MD Lipofectamine 2000 Life Technologies,Carlsbad, CA AF488 anti-mouse IgG1 isotype Biolegend, San Diego, CAAF488 anti-human GITR Ebioscience, San Diego, CA AF647 anti-human NGFRBD, San Jose, CA APC anti-human CD137 BD, San Jose, CA APC anti-mouseIgG1 isotype Biolegend, San Diego, CA Clone Manager v9 software Sci-EdSoftware, Cary, NC Fetal bovine serum (FBS), heat inactivated LifeTechnologies, Carlsbad, CA LTβR (TNFRSF3) pCMV6-XI.4 expression vectorOrigene Technologies, Inc., Rockville, MD MaxiSorp 96 well flat bottomplate VWR, Radnor, PA Newborn calf serum, heat inactivated LifeTechnologies, Carlsbad, CA NGFR (TNFRSF16) pCMV6-XL5 expression vectorOrigene Technologies, Inc., Rockville, MD PE anti-human HVEMEbioscience, San Diego, CA PE anti-human LtβR R&D systems, Minneapolis,MN PE anti-human TNFRSF 1B BD, San Jose, CA PE anti-mouse IgG1 isotypeBiolegend, San Diego, CA PE anti-rat isotype Ebioscience, San Diego, CAPBS, pH 7.2 without calcium and magnesium Life Technologies, Carlsbad,CA Recombinant human TNFRSF1β (TNFRII) R&D systems, Minneapolis, MNRecombinant human TNFRSF3 (LTβR) R&D systems, Minneapolis, MNRecombinant human TNFRSF4 (OX40) R&D systems, Minneapolis, MNRecombinant human TNFRSF5 (CD40) R&D systems, Minneapolis, MNRecombinant human TNFRSF6 (FAS) R&D systems, Minneapolis, MN Recombinanthuman TNFRSF9 (CD137) In house protein; lot# AMPur19 Recombinant humanTNFRSF11A (RANK) R&D systems, Minneapolis, MN Recombinant humanTNFRSF11B (OPG) R&D systems, Minneapolis, MN Recombinant human TNFRSF14(HVEM) R&D systems, Minneapolis, MN Recombinant human TNFRSF16 (NGFR)R&D systems, Minneapolis, MN Recombinant human TNFRSF18 (GITR) In houseprotein; lot# LBPur0025 Recombinant human TNFRSF21 (DR6) R&D systems,Minneapolis, MN TNFRSF1β pCMV6-XL5 expression vector OrigeneTechnologies, Inc., Rockville, MD Tween-20 Sigma-Aldrich, St. Louis, MO3.2 Methods3.2.1 Search for Proteins with Close Sequence Homology to Human OX40

In order to identify human proteins with amino acid sequence identity tohuman OX40, a protein sequence homology Basic Local Alignment SearchTool (BLASTP) search was conducted using the protein sequence of OX40(CCDS 11/UniProt P43489). Nineteen TNFRSF family members/isoforms wereidentified. The full-length sequences of these proteins were verifiedusing both the CCDS and UniProt databases (www.uniprot.org). CloneManager version 9 software was used to perform an assembled alignmentagainst the human OX40 reference using a blosum62 scoring matrix todetermine the percentage of amino acid identity between human OX40 andthe proteins identified in the BLASTP search (Table 3-2).

3.2.2 Binding Specificity of OX40mAb24 for OX40 Relative to Other TNFRSFMembers Expressed in HEK293 Cells

cDNA constructs capable of directing the expression of individual TNFRSFmembers, when transfected into mammalian cells, were obtained fromOrigene Technologies, Rockville, Md. These cDNA constructs wereamplified and purified by the Protein Sciences group at MedImmune inGaithersburg, Md. for use in transient transfections. For individualexpression of each of the TNFRSF members, HEK293 cells were transfectedusing Lipofectamine 2000 (Life Technologies, Carlsbad, Calif.) combinedwith 0.5 μg DNA of an expression vector encoding one of the TNFRSFmembers, according to the manufacturer's suggested protocol forLipofectamine 2000. Forty-eight hours post transfection, cells wereremoved from tissue culture plates by trypsinization. Trypsin wasneutralized by the addition of serum-containing complete mediumRPMI-1640 plus 10% FBS), followed by cell pelleting and washes incomplete medium. Cells were then suspended in cold FACS buffer (PBS+2%FBS) and plated into 96 well non-tissue culture-treated plates forbinding studies with TNFRSF member-specific mAbs (Table 3-3) andOX40mAb24.

For binding of antibodies or OX40mAb24, HEK cells were pelleted, FACSbuffer removed, and cells suspended in FACS buffer containing 2 μg/mLpropidium iodide (PI) and either a fluorochrome-labeled mAb, specificfor the transfected TNFRSF member, at a concentration recommended by themanufacturer or with OX40mAb24 at a concentration of 1 μg/mL. Forbinding controls, cells were separately incubated withfluorochrome-labeled isotype control antibodies. Cells were incubatedwith antibodies for 1 hour at 4° C. in the dark. Thereafter, cellsincubated with fluorochrome-labeled monoclonal antibodies were washed incold FACS buffer and then binding events collected and analyzed by flowcytometry using an LSRII flow cytometer (BD Biosciences, San Jose,Calif.) and FlowJo software, as described in Section 3.2.3. Cellsincubated with OX40mAb24 were washed in ice cold FACS buffer and thensuspended 25 μg/mL of Alexa Fluor® 647 goat antihuman IgG (H+L)secondary antibody and incubated for a further 30 minutes at 4° C. inthe dark. For a secondary antibody binding control cells were incubatedin the absence of OX40mAb24, but in the presence of fluorochrome-labeledsecondary antibody alone. Thereafter, cells were washed and suspended incold FACS buffer for analysis on an LSRII flow cytometer.

3.2.3 Flow Cytometry Analysis

Flow cytometry standard (FCS) data was examined using FlowJo software(Ashland, Oreg.). To analyze mAb binding, cells were first gated forviable (PI negative) cells, and then the mean fluorescence intensity(MFI) of events was plotted versus total number of events to generatebinding histograms. The geometric MFI of all viable cells was determinedfor each sample so that the fold MFI over background (isotype control orsecondary antibody alone) could be determined.

3.2.4 OX40mAb24 Binding Specificity ELISA

An eight point, two-fold dilution series of each recombinant humanTNFRSF protein was prepared after dilution of stock proteins to 5 μg/mLin PBS. Subsequently, 50 μL of each antigen dilution was transferred induplicate to wells of a Nunc 96-well MaxiSorp flat bottom plate, andincubated overnight at 4° C. to adsorb proteins to the plate.Afterwards, plates were washed three times with PBS in a BioTek® platewasher to remove unbound protein. Anti OX40 mAb29, 9B12, and controlantibodies were diluted in PBS to a final concentration of 10 μg/mL, and50 μL of mAb were added to each well and incubated at room temperaturefor one hour to bind mAb to plate-bound protein. Thereafter, wells werewashed with PBS/Tween-20 0.1% (volume/volume) using a BioTek® platewasher. HRP conjugated goat anti-human or goat anti-mouse secondaryantibodies at 10 μg/mL was added to each well and incubated at roomtemperature for 1 hour. After three washes in PBS/Tween-20 0.1%, 50 μLof TMB substrate was added to each well and incubated at roomtemperature for 5 minutes to develop the colorimetric product. Reactionswere stopped by adding 50 μL of 0.5 molar H₂SO₄ to wells, and platesread immediately at 450 nm using an Envision C plate reader fordetection of the colorimetric product. Results were graphed in GraphPadPrism software for Windows, version 5.01, and binding curves generatedusing non-linear regression analysis for single site binding.

3.3 Results

The protein sequence homology BLASTP search on human OX40 identified 19human TNFRSF proteins or isoforms that shared 15-27% amino acid sequenceidentity with the full-length OX40 sequence. The proteins and theirpercentages of sequence identity are listed in Table 3-2.

TABLE 3-2 Amino Acid Sequence Identity of Twelve TNFRSF Members with theHighest Homology to Human OX40. % identity UniProt protein TNFRSF memberAlternate names with OX40 sequence ID TNFRSF11A RANK, CD265 27 Q9Y6Q6iso 2 (delta 7, 8, 9) TNFRSF6B DcR3 25 O95407 TNFRSF18 GITR, AITR 25Q9Y5U5 iso 2 TNFRSSF10C DCR1, TRAIL-R3 24 O14798 TNFRSF5 CD40 23 P25942iso 2 TNFRSF18 GITR, AITR 23 Q9Y5U5 iso 3 TNFRSF18 GITR, AITR 23 Q9Y5U5TNFRSF9 CD137, 4-1BB 22 Q07011 TNFRSF5 CD40 21 P25942 TNFRSF14 TR2,HVEM-A 21 Q92956 TNFRSF16 NGF receptor 21 P08138 TNFRSF3 LTβR, TNFRIII20 P36941 TNFRSF6 Fas 20 P25445 iso 6 Tmdcl (A) TNFRSF6 Fas 20 P25445TNFRSF3 LTβR, TNFRIII 19 P36941 iso 2 TNFRSF6 Fas 19 P25445 iso 7FasExo8Del TNFRSF11B Osteoprotegerin 18 O0030 TNFRSF1B TNFR1b, TNFR2, 18P20333 CD120b TNFRSF11A RANK, CD265 16 Q9Y6Q6 CCDS = consensus codingsequence; ID = identifier; NA = not applicable; TNFRSF = tumor necrosisfactor receptor superfamily; UniProt—Universal Protein Resource

Binding of OX40mAb24 to transiently transfected HEK293 cells thatexpressed human NGFR, LTBR, TNFR2, GITR, CD137 or HVEM was assessed byflow cytometry as described in Section 3.1.3 above. Cell surfaceexpression of human NGFR, LTBR, TNFR2, GITR, CD137 and HVEM wasconfirmed using commercially available antibodies specific for eachhuman TNFRSF protein; fold increase in MFI compared to isotype controlantibodies for each TNFRSF protein is shown in Table 3-3. Binding ofOX40mAb24 to HEK293 cells that expressed human NGFR, LTBR, TNFR2, GITR,CD137 or HVEM was not substantially above that seen for binding of thesecondary antibody alone to those same cells (Table 3-3, FIG. 4A). Incontrast, binding of OX40mAb24 to a Jurkat cell line that constitutivelyoverexpresses OX40 was 48-fold greater, by mean MFI, than the binding offluorochrome labeled secondary antibody alone (Table 3-3 and FIG. 4B).

TABLE 3-3 Fold Binding of Fluorochrome-labeled TNFRSF-specificMonoclonal Antibodies and OX40mAb24 to TNFRSF-overexpressing HEK293Cells or OX40mAb24 to OX40-overexpressing Jurkat Cells. Cell LineReceptor-Specific OX40mAb24 Transferred Commercial binding (ratio TNFRSFwith mAb binding to secondary member TNFRSF (ratio to isotype antibodyexpressed Member control MFI) alone MFI) OX40 Jurkat ND 48 TNFRSF16(NGFR) HEK293 17 2.2 TNFRSF3 (LTβR) HEK293 146 1.0 TNFRSF1β HEK293 5.01.0 TNFRSF18 (GITR) HEK293 37 1.1 TNFRSF9 (CD137) HEK293 24 1.2 TNFRSF14(HVEM) HEK293 69 1.1 mAb = monoclonal antibody; MFI = mean fluorescenceintensity; ND = not determined; TNFRSF = tumor necrosis factor receptorsuperfamily

In an ELISA format, binding of an antibody containing the Fab arms ofOX40mAb24 but with IgG1 Fc domain containing three amino acidmodifications (mAb29) was specific for OX40 (FIG. 5A), showing nospecific binding above background to recombinant human NGFR (TNFRSF16),LTβR (TNFRSF3), TNFR2 (TNFRSF1β), GITR (TNFRSF18), CD137 (TNFRSF9), HVEM(TNFRSF14), DR6 (TNFRSF21), osteoprotegerin (OPG; TNFRSF11B), RANK(TNFRSF11A), FAS (TNFRSF6), and CD40 (TNFRSF5). 9B12, the mouseanti-human OX40 IgG1 monoclonal antibody that was “humanized” to createOX40mAb24, demonstrated similar lack of binding to these TNFRSF proteins(FIG. 5B).

3.4: Conclusions

Binding of OX40mAb24 and 9B12 to human OX40 is specific, and do notcross-react with highly related TNFRSF proteins.

Example 4 Ability of OX40mAb24 to Co-stimulate Primary Human CD4⁺ TCells Through OX40 In Vitro

In this example, the ability of OX40mAb24 to enhance activation of Tcells, in combination with activation through the CD3/T cell receptor(TCR) complex, was assessed using a plate-based human CD4⁺ T cellproliferation and cytokine release assay. Soluble OX40mAb24 activity wasalso examined, as were the activity of soluble and plate-bound OX40mAb24in the absence of CD3/TCR signaling.

4.1 Materials

Materials used in this study are listed in Table 4-1.

TABLE 4-1 Materials Item Source AlexaFluor ® 647 goat anti-human IgG(H + L) Life Technologies, Carlsbad, CA Anti-human CD4 EFluor450 ®eBioscience, San Diego, CA Bovine serum albumin (BSA) Sigma, SaintLouis, MO CFSE cell labeling kit Life Technologies, Carlsbad, CAComplete RPMI medium: RPMI-1640 + 10% FBS Materials from LifeTechnologies, Carlsbad, CA Deep well plates, polypropylene, 2 mL VWR,Radnor, PA EasySep CD4⁺ T cell enrichment kit Stem Cell Technologies,Vancouver, BC Canada FlowJo software TreeStar, Ashland, OR Goatanti-human IgG, Fcγ-specific Jackson ImmunoResearch, West Grove, PA Goatanti-mouse IgG, Fcγ-specific Jackson ImmunoResearch, West Grove, PA Heatinactivated newborn calf serum Life Technologies, Carlsbad, CA IL-2,recombinant human Preprotech, Rocky Hill, NJ Leuko Pak AllCells,Alameda, CA LSM MP Biomedicals, Santa Ana, CA LSR II flow cytometer BDBiosciences, San Jose, CA Mouse anti-human CD3 antibody clone OKT3Biolegend, San Diego, CA Newborn calf serum, beat inactivated (FBS) LifeTechnologies, Carlsbad, CA Non-issue culture treated round-bottom 96well plates VWR, Radnor, PA PHA-L Roche Applied Science, Indianapolis,IN Phosphate Buffered Saline (PBS) pH 7.2 without Life Technologies,Carlsbad, CA Calcium and Magnesium Prism software, v 5.01 GraphpadSoftware, San Diego, CA Propidium iodide (1 mg/mL solution) Sigma, SaintLouis, MO RosetteSep CD4⁺ T cell enrichment kit StemCell Technologies,Vancouver, BC Canada RPMI-1640 Life Technologies, Carlsbad, CA Th1/Th2multi-cytokine detection array Mesoscale Discovery (MSD), Rockville, MDVi-Cell counter Beckman Coulter, Indianapolis, IN Whole blood, sodiumheparin anti-coagulated MedImmune Blood Donor Program, Gaithersburg, MD4.2 Assays4.2.1 Plate-immobilized Bioactivity of OX40mAb24

The bioactivity of OX40mAb24 was determined by measurement of human CD4⁺T cell proliferation and cytokine production in a plate-based drugcapture assay (FIG. 6).

Enriched human CD4⁺ T cells were isolated from healthy donor whole bloodusing a RosetteSep CD4⁺ T cell enrichment kit, according to themanufacturer's protocol. Assays were performed with cells from fourindependent donors.

CD4⁺ T cells were suspended in complete RPMI culture medium and the cellconcentration adjusted to 1.0×10⁶ per mL. Final concentrations of 2μg/mL phytohemagglutinin-leucoagglutinin (PHA-L) and 20 IU/mLrecombinant human IL-2 were added, and cells were cultured at 37° C. and5% CO₂ in a humidified tissue culture incubator for 2 days to activate Tcells and up-regulate OX40.

Non-tissue culture treated round-bottom 96 well assay plates were coatedwith 100 μL of 2 μg/mL goat anti-mouse Fcγ-specific IgG and 2 μg/mL goatanti-human Fcγ-specific IgG in PBS. Goat anti-human IgG captureantibodies were not added to wells intended for assay of solubleOX40mAb24 activity. Plates were incubated overnight at 4° C., washedwith 200 μL of PBS, and blocked for 90 minutes at 37° C. with 1% BSA inPBS (1% BSA/PBS). The plates were washed with PBS and 2 ng/mL of mouseanti-human CD3 clone OKT3 reconstituted in 1% BSA/PBS was added to theplates for 90 minutes at 37° C. The plates were washed with PBS toremove unbound OKT3, OX40mAb24, R347 human IgG1 control mAb, 9B12, andmouse IgG1 control mAb clone MOPC-21 were each reconstituted in 1%BSA/PBS starting at 0.918 μg/mL (3.0 nM) and serially diluted over a3-fold dilution series and then added to assay plates and incubated for90 minutes at 37° C.

Activated primary human CD4⁺ T cells were collected, washed in completeRPMI medium, and the concentration adjusted to 1.0×10⁶ viable cells/mL.Cells were labelled with carboxyfluorescein succinimidyl ester (CFSE),according to the manufacturer's instructions, with the exception ofusing 1.25 μM CFSE as opposed to the recommended 5 μM with an incubationof 10 minutes at 37° C. After labeling, cells were suspended in completeRPMI medium and the concentration was adjusted to 0.5×10⁶ per mL. Theplates were washed with PBS and 200 μL of CD4⁺ T cells (100,000/well)were added to each well. For wells containing soluble OX40mAb24,OX40mAb24 was diluted in complete RPMI medium to the highest finalconcentrations used for plate-bound OX40mAb24. Cells in the plate werepelleted by centrifugation at 380×g and incubated at 37° C. for 3 days.After 72 hours incubation time, 40 μL of cell culture supernatant wasremoved for cytokine release measurement. CD4⁺ T cells were pelleted,and washed once with PBS containing 2% FBS (FACS buffer). Cells weresuspended in binding mix containing anti-human CD4 eFluor450® labeledantibody for identification of CD4⁺ T cells, and propidium iodide (PI)for live/non-viable cell discrimination, and incubated for 30 minutes.Following incubation, cells were washed in FACS buffer, re-suspended inFACS buffer and analyzed by flow cytometry using an LSRII flow cytometerand FlowJo software for analysis of Flow Cytometry Standard (FCS)formatted data.

To analyze T cell proliferation, live (PI negative) events were gatedusing FlowJo software, and the percentage of CD4-gated cells showingdilution of CFSE determined as a measure of the percentage of cellsundergoing proliferation.

To analyze cytokine release, cell culture supernatants obtained after 72hours of culture were measured for cytokine content using a 10-plexhuman Th1/Th2 cytokine analysis kit from MesoScale Discovery(Gaithersburg, Md.) according to the manufacturer's protocol. This kitemploys an electrochemical detection method to quantitatively measurethe following human cytokines: IFNγ, IL-2, IL4, IL-5, IL-8, IL-10, IL-12p70, IL-13, and IL-1β.

GraphPad Prism version 5.01 for Windows, GraphPad Software, San DiegoCalif. USA, www.graphpad.com was used to plot the log of mAbconcentration versus either proliferation or cytokine release values.The effective concentrations resulting in 20%, 50% and 90% maximaleffect (EC₂₀, EC₅₀, and EC₉₀) values for OX40mAb24 bioactivity werecalculated from sigmoidal dose-response bioactivity curves using theECAnything function.

4.3 Results

Proliferation data from each of the four donors and cytokine releasedata from one donor are shown in FIGS. 7A-C and FIGS. 8A-E. EC₂₀, EC₅₀,and EC₉₀ potency values for human primary CD4⁺ T cell proliferation areshown in Table 4-2; potency values for human primary CD4⁺ T cellcytokine release assays in Table 4-3 through Table 4-7; Meanproliferation and cytokine release values for OX40mAb24 and 9B12 areshown in Table 4-8 and Table 4-9, respectively.

OX40mAb24 co-stimulated proliferation of primary human CD4⁺ T cells(n=4) in a concentration-dependent manner with EC₂₀, EC₅₀, and EC₉₀values of 21, 28, and 72 pM, respectively. 9B12 co-stimulatedproliferation of CD4⁺ T cells with EC₂₀, EC₅₀, and EC₉₀ values of 106,218, and 622 pM. Therefore, the ratio of 9B12 to OX40mAb24concentrations required to induce a 50% of maximum proliferativeresponse was 8 to 1 (Table 4-2).

OX40mAb24 and 9B12 co-stimulated primary human CD4⁺ T cells to releasecytokines (n=4). Mean EC₂₀, EC₅₀, and EC₉₀ values were less potent thanvalues for proliferation, and are summarized in Table 4-8 and Table 4-9.Non-linear regression analysis could not be conducted for IL-2, IL-4,IL-8, IL-12 p70, and IL-1β assay results for both mAbs, due to poorlyformed or non-existent sigmoidal dose-response curves.

TABLE 4-2 Mean EC₂₀, EC₅₀ and EC₉₀ Values for OX40mAb24 and 9B12 in aPrimary Human CD4⁺ T cell Proliferation Assay Activity in absence ofDonor Monoclonal EC₂₀ EC₅₀ EC₉₀ Soluble TCR Number Antibody (95% CI), pM(95% CI), pM (95% CI), pM activity stimulation 367 OX40mAb24   32 (22,46)   38 (20, 72)  71 (17, 29) Not Tested None 661 OX40mAb24   33 (28,39)   51 (33, 77) 128 (96, 172) Not Tested None 645 OX40mAb24  7.5 (2.7,21)  8.3 (2.8, 25)  35 (7.1, 173) None Not Tested 651 OX40mAb24  9.6(6.3, 15)   16 (9.1, 26)  54 (30, 99) None Not Tested Mean OX40mAb24  21 (6.9)   28 (9.8)  72 (20) (Standard Error of the Mean), pM 367 9B1299.8 (80.0, 125)  156 (90.2, 269) 337 (214, 530) Not Tested Not Tested661 9B12  128 (113, 144)  237 (164, 342) 535 (464, 617) Not Tested NotTested 645 9B12 77.5 (30.2, 199)  169 (67.0, 425) 686 (152, 3080) NotTested Not Tested 651 9B12  118 (80.8, 173)  309 (201, 475) 929 (511,1690) Not Tested Not Tested Mean 9B12  106 (11.1)  218 (35.2) 622 (125)(Standard Error of the Mean), pM CI = confidence interval; EC₂₀ =effective concentration resulting in 20% of maximal effect; EC₅₀ = halfmaximal effective concentration; EC₉₀ = effective concentrationresulting in 90% of maximal effect; TCR = T cell receptor.

TABLE 4-3 Mean EC₂₀, EC₅₀ and EC₉₀ Values for IFNγ Induced by OX40mAb24or 9B12 in a Primary Human CD4⁺ T Cell Bioactivity Assay Activity inabsence of Donor Monoclonal EC₂₀ EC₅₀ EC₉₀ Soluble TCR Number Antibody(95% CI), pM (95% CI), pM (95% CI), pM activity stimulation 367OX40mAb24 38.6 (21.3, 70.0)  57.2 (31.2, 105)  106 (34.6, 328) NotTested None 661 OX40mAb24 ND ND ND Not Tested None 645 OX40mAb24 32.1(20.8, 49.7)  58.2 (42.0, 80.8)  150 (77.6, 289) None Not Tested 651OX40mAb24 47.5 (24.5, 91.8)  77.4 (51.4, 116)  168 (86.7, 324) None NotTested Mean OX40mAb24 39.4 (4.46)  64.3 (6.57)  141 (18.4) (StandardError of the Mean), pM 367 9B12 ND ND ND Not Tested Not Tested 661 9B12ND ND ND Not Tested Not Tested 645 9B12  706 (518, 963)  2380 (1420,3970) 16300 (5480, 48300) Not Tested Not Tested 651 9B12  344 (274, 430)  758 (639, 900)  2660 (780, 3990) Not Tested Not Tested Mean 9B12  525(148)  1570 (662)  9480 (5569) (Standard Error of the Mean), pM CI =confidence interval; EC₂₀ = effective concentration resulting in 20% ofmaximal effect; EC₅₀ = half maximal effective concentration; EC₉₀ =effective concentration resulting in 90% of maximal effect; TCR = T cellreceptor.

TABLE 4-4 Mean EC₂₀, EC₅₀ and EC₉₀ Values for TNFα Induced by OX40mAb24or 9B12 in a Primary Human CD4⁺ T cell Bioactivity Assay Activity inabsence of Donor Monoclonal EC₂₀ EC₅₀ EC₉₀ Soluble TCR Number Antibody(95% CI), pM (95% CI), pM (95% CI), pM activity stimulation 367OX40mAb24 ND ND ND Not Tested None 661 OX40mAb24 ND ND ND Not TestedNone 645 OX40mAb24 37.6 (22.5, 62.7) 54.1 (29.1, 101)  96.2 (26.7, 347)None Not Tested 651 OX40mAb24 ND ND ND None Not Tested Mean OX40mAb24 NDND ND (Standard Error of the Mean), pM 367 9B12 ND ND ND Not Tested NotTested 661 9B12 ND ND ND Not Tested Not Tested 645 9B12  306 (258, 503) 670 (527, 853)  1800 (1100, 2920) Not Tested Not Tested 651 9B12  388(300, 502)  764 (640, 919)  2260 (1500, 3410) Not Tested Not Tested Mean9B12  347 (33.5)  717 (38.4)  2030 (188) (Standard Error of the Mean),pM CI = confidence interval; EC₂₀ = effective concentration resulting in20% of maximal effect; EC₅₀ = half maximal effective concentration; EC₉₀= effective concentration resulting in 90% of maximal effect; ND = notdetermined due to poor curve fit (EC₂₀, EC₅₀, EC₉₀) or lack ofsufficient in values to determine mean and standard error of the mean;TCR = T cell receptor.

TABLE 4-5 Mean EC₂₀, EC₅₀ and EC₉₀ Values for IL10 Induced by OX40mAb24or 9B12 in a Primary Human CD4⁺ T Cell Bioactivity Assay Activity inabsence of Donor Monoclonal EC₂₀ EC₅₀ EC₉₀ Soluble TCR Number Antibody(95% CI), pM (95% CI), pM (95% CI), pM activity stimulation 367OX40mAb24 41.6 (17.8, 97.0)  59.5 (25.4, 139)  105 (25.0, 440) NotTested None 661 OX40mAb24 63.5 (29.6, 136)  89.5 (64.2, 125)  154 (93.3,255) Not Tested None 645 OX40mAb24 ND ND ND None Not Tested 651OX40mAb24 53.0 (31.0, 90.6)  86.4 (65.0, 115)  188 (110, 321) None NotTested Mean OX40mAb24 52.7 (6.32)  78.5 (9.53)  149 (24.1) (StandardError of the Mean), pM 367 9B12  130 (83.3, 204)   198 (139, 284)  385(224, 662) Not Tested Not Tested 661 9B12 ND ND ND Not Tested Not Tested645 9B12  528 (363, 767)  1220 (858, 1740) 4630 (2050, 10400) Not TestedNot Tested 651 9B12  405 (300, 547)   796 (649, 976) 2320 (1440, 3730)Not Tested Not Tested Mean 9B12  354 (118)   738 (296) 2445 (1227)(Standard Error of the Mean), pM CI = confidence interval; EC₂₀ =effective concentration resulting in 20% of maximal effect; EC₅₀ = halfmaximal effective concentration; EC₉₀ = effective concentrationresulting in 90% of maximal effect; ND = not determined due to poorcurve fit (EC₂₀, EC₅₀, EC₉₀) or lack of sufficient in values todetermine mean and standard error of the mean; TCR = T cell receptor.

TABLE 4-6 Mean EC20, EC50 and EC90 Values for IL13 Induced by OX40mAb24or 9B12 in a Primary Human CD4⁺ T Cell Bioactivity Assay Activity inabsence of Donor Monoclonal EC₂₀ EC₅₀ EC₉₀ Soluble TCR Number Antibody(95% CI), pM (95% CI), pM (95% CI), pM activity stimulation 367OX40mAb24 ND ND ND Not Tested None 661 OX40mAb24 ND ND ND Not TestedNone 645 OX40mAb24  44.7 (26.5, 75.5)  73.8 (52.2, 104)  163 (93.3, 286)None Not Tested 651 OX40mAb24  36.2 (24.9, 52.7)  65.0 (49.6, 85.3)  164(99.2, 273) None Not Tested Mean OX40mAb24  40.5 (4.25)  69.4 (4.40) 164 (0.50 (Standard Error of the Mean), pM 367 9B12 ND ND ND Not TestedNot Tested 661 9B12   154 (841, 283)   238 (166, 341)  472 (278, 800)Not Tested Not Tested 645 9B12  1100 (413, 2910)  5450 (1060, 2800) 6930 (4310, 1110000) Not Tested Not Tested 651 9B12   495 (324, 756) 1770 (896, 3510) 13400 (2980, 60400) Not Tested Not Tested Mean 9B12  583 (277)  2486 (1550)  6930 (3730) (Standard Error of the Mean), pMCI = confidence interval; EC₂₀ = effective concentration resulting in20% of maximal effect; EC₅₀ = half maximal effective concentration; EC₉₀= effective concentration resulting in 90% of maximal effect; ND = notdetermined due to poor curve fit (EC₂₀, EC₅₀, EC₉₀) or lack ofsufficient in values to determine mean and standard error of the mean;TCR = T cell receptor.

TABLE 4-7 Mean EC₂₀, EC₅₀ and EC₉₀ Values for IL5 Induced by OX40mAb24or 9B12 in a Primary Human CD4⁺ T cell Bioactivity Assay Activity inabsence of Donor Monoclonal EC₂₀ EC₅₀ EC₉₀ Soluble TCR Number Antibody(95% CI), pM (95% CI), pM (95% CI), pM activity stimulation 367OX40mAb24 33.2 (21.2, 52.1)  46.7 (24.7, 88.0)  79.8 (12.3, 519) NotTested None 661 OX40mAb24 43.4 (27.6, 68.4)  69.0 (49.7, 95.7)   144(86.8, 238) Not Tested None 645 OX40mAb24 ND ND ND None Not Tested 651OX40mAb24 56.2 (35.5, 88.9)  95.9 (76.1, 121)   224 (128, 391) None NotTested Mean OX40mAb24 44.3 (6.65)  70.5 (14.2)   149 (41.7) (StandardError of the Mean), pM 367 9B12 ND ND ND Not Tested Not Tested 661 9B12ND ND ND Not Tested Not Tested 645 9B12 ND ND ND Not Tested Not Tested651 9B12  423 (285, 627)  1090 (742, 1600)  4870 (1900, 12500) NotTested Not Tested Mean 9B12 ND ND ND (Standard Error of the Mean), pM CI= confidence interval; EC₂₀ = effective concentration resulting in 20%of maximal effect; EC₅₀ = half maximal effective concentration; EC₉₀ =effective concentration resulting in 90% of maximal effect; ND = notdetermined due to poor curve fit (EC₂₀, EC₅₀, EC₉₀) or lack ofsufficient in values to determine mean and standard error of the mean;TCR = T cell receptor.

TABLE 4-8 Summary of Mean EC₂₀, EC₅₀ and EC₉₀ Values for Proliferationand Cytokine Release for OX40mAb24 EC₂₀ (StdErr), EC₅₀ (StdErr), EC₉₀(StdErr), Bioactivity Readout pM pM pM CD4 T cell 21 (6.9) 28 (98) 72(20) proliferation IFNγ release 39.4 (4.46) 64.3 (6.57) 141 (18.4) TNFαrelease ND ND ND IL10 52.7 (6.32) 78.5 (9.53) 149 (24.1) IL13 40.5(4.25) 69.4 (4.40) 164 (0.50) IL5 44.3 (6.65) 70.5 (14.2) 149 (41.7)EC₂₀ = effective concentration resulting in 20% of maximal effect; EC₅₀= half maximal effective concentration; EC₉₀ = effective concentrationresulting in 90% of maximal effect; ND = not determined due toinsufficient n value to calculate a mean and standard error of the mean;StdErr = standard error of the mean.

TABLE 4-9 Summary of Mean EC₂₀, EC₅₀ and EC₉₀ Values for Proliferationand Cytokine Release for 9B12 EC₂₀ (StdErr), EC₅₀ (StdErr), EC₉₀(StdErr), Bioactivity Readout pM pM pM CD4 T cell  106 (11.1)  218(35.2) 622 (125) proliferation IFNγ release 525 (148) 1570 (662)  9480(5569) TNFα release  347 (33.5)  717 (38.4) 2030 (188)  IL10 354 (118)738 (296) 2445 (1227) IL13 583 (277) 2486 (1547) 6934 (3732) IL5 ND NDND EC₂₀ = effective concentration resulting in 20% of maximal effect;EC₅₀ = half maximal effective concentration; EC₉₀ = effectiveconcentration resulting in 90% of maximal effect; ND = not determineddue to insufficient n value to calculate a mean and standard error ofthe mean; StdErr = standard error of the mean.

The activity of non-plate bound, soluble OX40mAb24 was determined.Soluble OX40mAb24 did not induce either primary human CD4⁺ T cellproliferation (FIG. 7A-C) or cytokine release (FIGS. 8A-E) above levelsobserved for either anti-CD3 antibody alone or in the presence of R347human IgG1 control mAb. Plate bound anti-CD3 antibody alone produced aminimal-to-moderate level of proliferation and cytokine release. Thelack of activity demonstrated here by soluble OX40mAb24 is in agreementwith the absence of activity observed for soluble OX40mAb24 withoutcell-based cross-linking in a 2-cell bioactivity assay that measuredOX40 mediated NFκB signaling (see Example 5 below).

Likewise, OX40mAb24, either immobilized on the plate surface or added asa soluble unbound protein, in the absence of a sub-mitogenic anti-CD3antibody signal induced little-to-no CD4⁺ T cell proliferation (FIGS.7A-C) or cytokine release (FIGS. 8A-E). These results demonstrated that,in this study, OX40mAb24 does not have activity in primary human CD4⁺ Tcells in the absence of simultaneous CD3/TCR ligation.

4.4 Conclusions

OX40mAb24 induced proliferation and cytokine release of primary humanCD4⁺ T cells in a concentration-dependent manner similar to that of theantibody from which it was humanized 9B12 (FIGS. 8A-E and FIGS. 9A-E).OX40mAb24 demonstrated activity as a plate-bound, but not as a soluble,protein. Furthermore, OX40mAb24 activity occured concurrent with CD3/TCRsignaling.

Example 5 Determination of the In Vitro Activity of OX40mAb24 in 2-cellBased Bioactivity Assays Using Jurkat NFκB-luciferase Reporter T Cells

In this example, the ability of OX40mAb24 and 9B12 to signal throughhuman OX40 was assessed using a set of two-cell reporter bioactivityassays. Measurement of T cell activation through OX40 co-stimulation wasaccomplished by using an OX40-overexpressing Jurkat NFκB-luciferase Tcell reporter line that produces luciferase in response to stimulationof the NFκB signaling pathway (FIG. 10). NFκB signaling has beenreported to occur downstream of OX40 engagement, and can correlate withother measures of T cell activation, such as proliferation and cytokinerelease (Croft M, et al., Immunol Rev. 229:173-91 (2009)). The amount ofluciferase, and thus T cell activation, was measured by adding aluciferase substrate to cell lysates and measuring light emitted by thereaction product using a luminometer. Bioactivity of OX40mAb24cross-linked using cells engineered to express different Fcγ receptorcomplements, as well as soluble, non-FcγR-crosslinked OX40mAb24 wasmeasured.

5.1 Materials

Materials used in this study are listed in Table 5-1.

TABLE 5-1 Materials Item Source CD45⁺ microbeads Miltenyi, San Diego, CACollagenase III Worthington Biochemical Corporation, Lakewood, NJ DNAseI, from b ovine pancreas Sigma, Saint Louis, MO EDTA, 0 5M pH 8.0 LifeTechnologies, Carlsbad, CA Envision luminescence reader Perkin Elmer,Waltham, MA Heat inactivated newborn calf Life Technologies, Carlsbad,CA serum (FBS) LS column Miltenyi, San Diego, CA MACS buffer: PBS + 0.5%Materials from Life Technologies, BSA + 2 mM EDTA Carlsbad, CA Non-TCtreated round-bottom VWR, Radnor, PA 96 well plates Prism v5.01 softwareGraphPad, San Diego, CA Steady-Glo Luciferase Assay Promega, Madison, WISolution ViCell counter Beckman Coulter, Indianapolis, IN5.2 Two-cell Bioactivity Assay5.2.1 Isolation of Primary Human CD45⁺ Cells

Primary human CD45⁺ cells were isolated from human tumors. Tumor sampleswere removed from transport media and placed in sterile petri dish.Hank's Buffered Salt Solution was added and visible necrotic tissue orany normal tissue from tumor sample was dissected. Tissue was mincedinto small pieces (˜1 mm) and placed in a 50 mL conical tube, andCollagenase III enzyme mix (250 IU/mL collagenase III, 3 mM CaCl₂, 315μg/mL DNAase 1) was added, mixed and incubated. The digested sample wasfiltered through a 70 micron filter and washed with MACS buffer.Dissociated cells were pelleted and the cell number and viability weredetermined using a ViCell counter. Cells were suspended in MACS bufferwith CD45 microbeads and incubated on ice. Cells were washed andre-suspended in MACS buffer for positive selection of CD45⁺ cells usingan LS column. Bead-bound CD45⁺ cells were eluted by removing the columnfrom the magnet and adding MACS buffer to the column. Cells werepelleted and re-suspended in complete RPMI medium and used inbioactivity assays as described above.

5.2.2 Assay Protocol

OX40mAb24 and 9B12 were tested for bioactivity using a 2-cellbioactivity assay. Human OX40-overexpressing Jurkat NFκB-luciferasereporter clone 64 (OX40 Jurkat reporter) was used to measure OX40agonism (NFκB activity). A second, FcγR-expressing cell line, was usedto mediate OX40mAb24 cross-linking, which clusters and activates OX40 onthe OX40 Jurkat reporter cells (FIG. 10). The FcγR-expressing cell linesused for cross-linking included the Raji human B cell lymphoma line,CD32A-expressing HEK293, CD32B-expressing HEK293 or CD45⁺ immune cellsisolated from primary human tumors.

To determine the soluble activity of OX40mAb24, bioactivity assays werealso conducted using either parental HEK293 cells, which are FcγR nulland therefore are unable to cross-link OX40mAb24, or in the absence ofcross-linking cells altogether.

Prior to use, OX40 Jurkat reporter cells were cultured in complete RPMImedium in a tissue culture incubator at a density of 0.5-1.5×10⁶ per mL.Cells were passaged the day prior to the bioassay at a final density of10⁶ cells per mL.

OX40 Jurkat reporter cells, FcγR-expressing cell lines, or HEK parentalcells were collected, and pelleted. To isolate CD45⁺ cells from primaryhuman tumors and normal adjacent tissues, tissues were dissociated andCD45⁺ cells isolated and re-suspended in complete RPMI medium for use inbioactivity assays, as described below.

OX40mAb24, 9B12 and various control antibodies were serially diluted3-fold in complete RPMI medium.

OX40 Jurkat reporter cells plus FcγR-expressing cells, or HEK parentalcells, were added to a 96 well plate at 100,000 cells per well.OX40mAb24, 9B12 or control antibodies were added to cells in a dilutionseries with a starting concentration of 10 μg/mL, and incubated at 37°C. in a tissue culture incubator.

After 16-24 hours incubation time, 100 μL reconstituted Steady-Gloluciferase assay solution (Promega, Madison, Wis.) was added to eachwell and mixed to lyse cells and then incubated to equilibrateluciferase signal. Steady-Glo/sample lysate (150 μL) was transferredfrom each well to a 96 well, white walled assay plate for detection andluminescence read using a Perkin Elmer Envision luminescence reader.

GraphPad Prism version 5.01 for Windows (GraphPad Software, San Diego,Calif.), was used to plot the concentration of OX40mAb24, 9B12, R347human IgG1, mouse IgG1 clone MOPC-21 (x-axis is log 10 of proteinconcentration) versus luminescence RLU (y-axis). The EC₂₀, EC₅₀, andEC₉₀ values for bioactivity were determined using ECAnything (ECf) forf=20, f=50 and f=90 from sigmoidal dose-response (variable slope)bioactivity curves).

5.3 Results of 2-Cell Bioactivity Assays

Results for the bioactivity of OX40mAb24 and control antibodies areshown in FIG. 11A-D, FIGS. 12A-D and Table 5-2.

In the presence of an FcγR-expressing cell line (e.g., CD32A-expressingHEK293, CD32B-expressing HEK293, Raji B cells, or CD45⁺ cells isolatedfrom primary human tumors), OX40mAb24 demonstrated potent stimulation ofOX40-overexpressing Jurkat NFκB reporter cells, as measured by NFκBpathway activation. In the absence of a second cell type or in thepresence of HEK293 cells lacking exogenously expressed FcγRs, OX40mAb24exhibited minimal reporter activity (FIG. 11A-D).

Potency values (EC₂₀, EC₅₀, and EC₉₀) for the two-cell bioassays aresummarized in Table 5-2. The mean EC₂₀, EC₅₀, and EC₉₀ values across allassays were 228, 751, and 5630 pM, respectively.

Results for the bioactivity of 9B12 and control antibodies are shown inFIGS. 12A-D and the potency values are summarized in Table 5-3. The meanEC₂₀, EC₅₀, and EC₉₀ values for all assays were 519, 2530, and 41100 pM,respectively. Therefore, the ratio of 2-cell bioactivity (EC₅₀) for 9B12relative to that of OX40mAb24 was calculated to be 3.4 to 1.

TABLE 5-2 Two-cell Bioactivity of OX40mAb24 Experiment EC₂₀ EC₅₀ EC₉₀Number FcγR-expressing cell* (95% CI) pM (95% CI) pM (95% CI) pM 1 Raji298 (236, 378) 1140 (982, 1340)  9850 (6500, 14300) 2 CD32A-expressingHEK  104 (78.8, 138) 437 (370, 517) 4250 (2900, 6240) 3 CD32A-expressingHEK  100 (79.5, 126) 322 (281, 370) 2060 (1530, 2780) 4 Raji 350 (259,467) 1040 (855, 1270) 5930 (3740, 9400) 5 CD32B-expressing HEK 90.6(75.4, 109) 270 (242, 301) 1520 (1200, 1920) 6 Raji 180 (109, 296)  796(577, 1100)  8410 (3820, 18500) 7 CD32B-expressing HEK 280 (198, 394) 899 (710, 1140) 5730 (3290, 9970) 8 CD32B-expressing HEK 237 (196, 286)671 (592, 762) 3510 (2630, 4670) 9 Raji  676 (443, 1030) 1320 (970,1800) 3820 (2000, 7320) 10 Raji 297 (127, 694) 1260 (666, 2390) 12500(2570, 60500) 11 CD32B-expressing HEK 144 (113, 184) 700 (598, 818) 8540 (5770, 12600) 12 CD45⁺ cells, NSCLC  33.6 (24.7, 45.8)  81.6(68.5, 97.3) 334 (236, 471)  13 CD45⁺ cells, RCC 92.2 (75.4, 113) 337(296, 383) 2630 (1940, 3550) 14 CD45⁺ cells, NSCLC 247 (207, 295) 772(688, 867) 4700 (3600, 6140) 15 CD45⁺ cells, RCC 351 (155, 798) 1410(782, 2530) 12700 (2960, 54500) 16 CD32A-expressing HEK 166 (145, 191)553 (507, 603) 3720 (3060, 4530) Mean (Standard Error 228 (38.9)    751(101)   5630 (937)    of the Mean) NSCLC = non-small cell lung cancer;RCC = renal cell carcinoma *As indicated, assays used Raji,CD32A-expressing HEK293, CD32B-expressing HEK293 cells, or CD45⁺ cellsisolated from primary human tumor samples with OX40-expressing JurkatNFκB-luciferase clone 64 reporter cells.

TABLE 5-3 Two-cell Bioactivity of 9B12 Experiment EC₂₀ EC₅₀ EC₉₀ NumberFcγR-expressing cell* pM pM pM 1 Raji 1110 (791, 1570)  9280 (4460,19300)  267000 (57900, 1230000) 2 CD32A-expressing HEK 74.1 (53.0, 104)338 (279, 410)  3760 (2440, 5790) 3 CD32A-expressing HEK 66.7 (43.3,103) 225 (175, 289)  1550 (914, 2630)  4 Raji 1040 (834, 1300) 5040(3880, 6550)  61200 (32200, 116000) 5 CD32B-expressing HEK 249 (206,303) 726 (641, 823)  3950 (2980, 5240) 6 Raji 548 (408, 736) 2700 (2070,3620)  33700 (17000, 66700) 7 CD32B-expressing HEK 585 (520, 658) 1770(1620, 1930) 10200 (8280, 12500) 8 CD32B-expressing HEK 518 (449, 598)1340 (1210, 1480) 6050 (4800, 7630) 9 Raji  872 (504, 1510) 3070 (1910,4950) 22600 (7000, 73100) 10 Raji  571 (212, 1540)  3330 (1080, 10200) 54400 (3400, 872000) 11 CD32B-expressing HEK 650 (458, 923) 2360 (1790,3100) 18200 (9200, 36000) 12 CD45⁺ cells, NSCLC  104 (76.3, 143) 223(185, 269)  741 (501, 1100) 13 CD45⁺ cells, RCC ND ND ND 14 CD45⁺ cells,NSCLC ND ND ND 15 CD45⁺ cells, RCC 356 (132, 964) 2420 (938, 6250)  50500 (4140, 616000) Mean (Standard Error 519 (96.2)    2530 (687)   41100 (19700)     of the Mean) ND = not determined; NSCLC = non-smallcell lung cancer; RCC = renal cell carcinoma *As indicated, assays usedRaji, CD32A-expressing HEK293, CD32B-expressing HEK293 cells, or CD45⁺cells isolated from primary human tumor samples with OX40-expressingJurkat NFκB-luciferase clone 64 reporter cells.5-4: Conclusions

OX40mAb24 and 9B12 mediate the activation of human T cells as measuredby the stimulation of the NFκB pathway in an OX40-overexpressing JurkatNFκB-luciferase reporter cell line. In the absence of cells that expressFcγRs capable of cross linking via the Fc domains of OX40mAb24, minimalreporter cell-line activity was measured. However, in a 2-cell systemcomprising cells that express FcγR that are capable of cross-linking themAb, and a Jurkat overexpressing NFκB-luciferase reporter line forreadout of T cell activation, OX40mAb24 and 9B12 mediated potent OX40activation with mean EC₅₀ values of 751 pM and 2530 pM, respectively.Therefore, the bioactivity of OX40mAb24 in the 2-cell assay system wassimilar to that of 9B12.

Example 6 Ability of OX40mAb24 to Trigger Effector Function

In this example OX40mAb24 was assessed with respect to its ability totrigger Fc effector function namely, the ability of OX40mAb24 to triggerhuman natural killer (NK) cell-mediated antibody-dependent cellularcytotoxicity (ADCC) against primary human CD4⁺ T cells expressing highlevels of OX40, or to bind C1q, a prerequisite for complement-dependentcellular cytotoxicity (CDC) by the classical complement pathway.Versions of OX40mAb24 containing either a human IgG4P Fc domain (mAb28),or a triple mutation in the IgG1 Fc domain that reduces Fcγ RIIIabinding (mAb29) were utilized to assess the contribution of theOX40mAb24 IgG1 Fc domain to mediate ADCC activity. Also, the effectorfunctions of OX40mAb24 were compared to that of 9B12, a mouse anti-humanOX40 IgG1 monoclonal antibody that was humanized to create OX40mAb24.The anti-CD20 antibody rituximab binds to B cells expressing CD20 andwas used as a positive control for ADCC experiments. Because primaryactivated human CD4⁺ T cells do not express CD20, a separate assay usingthe Toledo B cell line, which does express CD20, was conducted tovalidate the activity of NK cells used in the ADCC assay system.

6.1 Materials

Materials used in this study are listed in Table 7-1.

TABLE 6-1 Materials Item Source Complement protein C1q Quidel, SanDiego, CA Complete RPMI medium: Materials from Life Technologies,RPMI-1640+ 10% FBS Carlsbad, CA DM-L medium Stem Cell Technologies,Vancouver, BC Canada GLC biosensor chip Bio-Rad, Hercules, CA FlowJoSoftware FlowJo, Ashland, OR IL-2, recombinant human Preprotech, RockyHill, NJ LSR II flow cytometer BD Biosciences, San Jose, CA Prismsoftware, v 5.01 Graphpad Software, San Diego, CA Propidium iodide (1mg/mL Sigma, Saint Louis, MO solution) ProteOn Manager 2.1 softwareBio-Rad, Hercules, CA ProteOn XPR36 instrument Bio-Rad, Hercules, CARosetteSep CD4 T cell Stem Cell Technologies, Vancouver, enrichment kitBC Canada RosetteSep Human NK cell Stem Cell Technologies, Vancouver,enrichment BC Canada ViCell counter Beckman Coulter, Indianapolis, INVybrant DiO cell labeling Life Technologies, Carlsbad, CA solution Wholeblood, sodium heparin MedImmune Blood Donor Program, anti-coagulatedMedImmune, Gaithersburg, MD6.2 Assays6.2.1 Antibody-dependent Cellular Cytotoxicity

The ADCC activity of OX40mAb24 relative to that of 9B12 and monoclonalantibodies containing the Fab arms of OX40mAb24 with either an IgG4P Fcdomain or a triple mutant (TM) human IgG1 Fc domain, was tested usingenriched primary human NK cells as effectors and OX40-expressing primaryhuman CD4⁺ T cells as targets. As a positive control, the activity ofeach NK cell preparation was tested using rituximab directed killing ofthe Toledo B cell line.

For the isolation of primary human CD4⁺ T cells, heparinanti-coagulated, whole blood obtained from healthy donors through theMedImmune Blood Donor Program was processed according to the followingprotocol: Stem Cell Technologies RosetteSep CD4 T cell isolation kitantibody mix (1 mL, Stem Cell Technologies, Vancouver, BC Canada) wasadded per 20 mL of whole blood, mixed, and incubated for 20 minutes (mm)at room temperature (RT). Blood was diluted 1:1 with sterile roomtemperature FACS buffer (PBS, pH 7.2 plus 2% heat inactivated newborncalf serum) and layered onto DM-L medium followed by centrifugation for20 min. After centrifugation, the buffy coat containing human CD4⁺ Tcells was removed and the cells were washed once with RT FACS buffer andonce with RT complete RPMI medium. Cells were counted on a ViCellcounter to determine cell number and viability and CD4⁺ T cells weresuspended in complete RPMI medium at a concentration of 1.0×10⁶ per mL.

Primary human CD4⁺ T cells (1.0×10⁶ per mL in complete RPMI medium) werecultured in a humidified tissue culture incubator at 37° C. and 5% CO₂for 48 hours with 2 μg/mL PHA-L and 20 IU/mL rhIL-2 to activate T cellsand up-regulate OX40 and were subsequently used in OX40mAb24 ADCCassays. All donors in the figures referenced below represent uniqueindividuals; that is, CD4⁺ T cells were not isolated from the same donorfor repeat ADCC experiments.

To discriminate target T cells, or cultured Toledo B cells, frompurified human NK cells in cytotoxicity assays, the fluorescent dye,DiO, was incorporated into the cell membrane of target cells using theVybrant DiO cell labeling solution (according to the manufacturer'sprotocol for suspension cell labeling). After activation and DiOlabeling of primary human CD4⁺ T cells and Toledo B cells, effector NKcells were isolated from sodium heparin anticoagulated blood from theMedImmune Blood Donor Program using the same protocol as above for humanCD4⁺ T cells, with the exception that 1 mL of RosetteSep NK cellisolation kit antibody mix was used in place of 1 mL of RosetteSep CD4⁺T cell isolation kit antibody mix. Isolated primary human NK cells werewashed two times with warm complete RPMI medium (RPMI-1640 plus 10%FBS), and then suspended in complete RPMI at a concentration of 2.67×10⁶per mL. Likewise, activated primary human CD4⁺ T cells were also washedtwo times in warm complete RPMI and suspended at a concentration of2.67×10⁵ per mL. Thereafter, 75 μL of primary human NK cells (200,000)and 75 μL of activated primary human CD4⁺ T cells (20,000) were added towells of sterile non-tissue culture treated round bottom 96-well platesfor an effector-to-target ratio of 10:1. In some experiments, completeRPMI medium (50 μL) containing OX40mAb24 or control mAb was added togive a final concentration of 10 μg/mL. In others, complete RPMI (50 μL)containing a 3-fold dilution series of OX40mAb24 or control mAbs,resulting in a final concentration of 67 nM, or a 27-fold dilutionseries of 9B12 or R347 human IgG1 starting from 67 nM, was added to theplated cells. Rituximab (10 μg/mL, control antibody) was used as apositive control in wells containing DiO labeled, CD20-expressing ToledoB cells, in place of OX40-expressing, activated primary human CD4⁺ Tcells, because activated CD4⁺ T cells do not express CD20. Cells in theADCC assay were gently pelleted by centrifugation at RT and subsequentlycultured for 24 hours.

At the end of the incubation period, cells were pelleted bycentrifugation and then suspended in FACS buffer containing 10 μg/mL ofpropidium iodide (PT) for flow cytometry analysis on a BD LSRII flowcytometer. Non-viable (PT positive) cells among DiO-positive target CD4⁺T cells or Toledo B cells were discriminated using FlowJo software afterfluorescence compensation.

Graphical representation of the data for NK cell ADCC assays, includingdetermination of mean values and standard error of the mean, wasgenerated using GraphPad Prism version 5.01 for Windows.

6.2.2 OX40mAb24 Binding to C1q

A ProteOn XPR36 instrument was used to determine the binding ofOX40mAb24 or 9B12 to human complement protein C1q purified from pooledhuman sera to OX40mAb24 or 9B12 by surface plasmon resonance. Standardamine coupling was used to immobilize OX40mAb24 or 9B12 to the surfaceof a GLC biosensor chip. Human C1q was suspended in PBS/0.005% Tween 20,pH 7.4 at five concentrations ranging from 26 nM to 1.6 nM. The sampleswere injected at 30 μL/min for 200 seconds, and the dissociation phasewas followed for 600 seconds. Sensorgram data was processed usingProteOn Manager 2.1 software (Bio-Rad) using 1:1 fitting.

6.3 Results

The ability of OX40mAb24, 9B12, and human IgG4P (mAb28) and IgG1-TM(mAb29) versions of OX40mAb24 to mediate ADCC was tested at saturatinglevels (10 μg/mL [67 nM]) for OX40 binding (see Example 2), using bothallogeneic and autologous mixtures of NK cells and OX40-expressingtarget cells (FIG. 13A and FIG. 14A). OX40mAb24 showed measurable ADCCactivity against activated human CD4⁺ T cells. In contrast, 9B12, mAb28and mAb29 showed no ADCC activity above that of the negative controls. Apositive-control antibody against human CD20 (rituximab) demonstratedADCC activity using the same NK cells, demonstrating that the NK cellswere all capable of robust ADCC activity (FIG. 13B and FIG. 14B).

In dose-response experiments, OX40mAb24 demonstratedconcentration-dependent ADCC activity against activated human CD4⁺ Tcells (FIGS. 15A-D, FIGS. 16A-D, FIGS. 17A-B, and Table 6-2. Incontrast, 9B12 did not show any detectable ADCC activity. In addition,the R347 human IgG1 control mAb did not show any detectable ADCCactivity, which confirms that ADCC activity mediated by OX40mAb24 wasdependent on engagement with OX40 on the target cells. Apositive-control antibody against human CD20 also demonstrated ADCCagainst a CD20-expressing B cell lymphoma cell line (FIG. 13B and FIG.14B); this supports the validity of the assay. Collectively, the resultsof these experiments confirmed that OX40mAb24 is capable of ADCC againsttarget cells that have been cultured under conditions previously shownto stimulate expression of cell surface OX40.

TABLE 6-2 Summary of OX40mAb24-mediated NK cell ADCC Potency ExperimentDonor EC20 EC50 EC90 Number Number (pM) (pM) (pM) 3 363 134 2380 2250003 504 309 1370 14600 4 464 37.0 3050 3330000 4 532 111 1310 66100 5 60154.4 501 16900 5 602 61.4 527 16000 Mean (standard error 118 (41.1) 1520(415) 611000 (545000) of the mean), pM

The potential for OX40mAb24 to bind to the human complement componentC1q was assessed using a surface plasmon resonance assay. In this assay,binding to C1q was used as a surrogate for activity in a complementdependent cytotoxicity assay (Dall'Acqua et al., J. Immunol177:1129-1138 (2006)). 9B12 was used as a negative control antibody.OX40mAb24 demonstrated a concentration-dependent ability to bind topurified human C1q protein (FIG. 18A). In contrast, 9B12 did notdemonstrate any detectable binding to C1q protein (FIG. 18B).

6.4 Conclusions

OX40mAb24 binds to C1q and triggers NK-mediated ADCC against activatedCD4⁺ T cells.

Example 7 In Vitro Comparability Studies with OX40mAb24 and Cynomolgusand Rhesus Monkey T Cells

In this example, the ability of OX40mAb24 to enhance activation of Tcells was determined using two-cell reporter bioactivity assays with aJurkat NFκB-luciferase T cell reporter line expressing cynomolgus(cyno)/rhesus monkey OX40. Fcγ receptor-mediated drug cross-linking wasmediated by either a rhesus B cell line, or by rhesus Fcγreceptor-expressing cells in a post-red blood cell lysis whole bloodsample. OX40 activation was measured as increased luciferase activity inresponse to stimulation of the NFκB signaling pathway downstream ofprimary human T cell activation. NFκB signaling is a well-studieddownstream event in OX40 signaling, and can correlate with othermeasures of T cell activation such as proliferation and cytokine release(Croft M, et al., Immunol Rev. 229:173-91 (2009)). In addition, theability of OX40mAb24 to enhance T cell receptor-mediated activation(co-stimulation) of rhesus T cells was tested using a plate-basedbioactivity assay in which CD4⁺ T cell proliferation was assessed. Anisotype control (NIP228 IgG1), was used to demonstrate specific OX40engagement.

In parallel, the bioactivity of 9B12, the murine anti-OX40 monoclonalantibody from which OX40mAb24 was derived, was measured to provide for acomparison of OX40mAb24 and 9B12 activity on non-human primate OX40.

7.1 Materials

Materials used in this study are listed in Table 7.1

TABLE 7-1 Materials Item Source Ammonium Chloride StemCell Technologies,Vancouver, BC Anti-human CD3 antibody, clone SP34-2 BD Biosciences, SanJose, CA Anti-human CD4-V450 antibody, clone L200 BD Biosciences, SanJose, CA Anti-human CD28 antibody, clone CD28.2 BD Biosciences, SanJose, CA CFSE cell labeling kit Life Technologies, Carlsbad, CAConcanavalin A Sigma, Saint Louis, MO Envision multilabel plate readerPerkin Elmer, Waltham, MA Goat anti-human Fcγ antibody JacksonImmunoresearch, West Grove, PA Goat anti-mouse Fcγ antibody JacksonImmunoresearch, West Grove, PA Heat inactivated fetal bovine serum LifeTechnologies, Carlsbad, CA Interleukin-2, recombinant human Preprotech,Rocky Hill, NJ LSR II flow cytometer BD Biosciences, San Jose, CALymphocyte separation medium (LSM) MP Biomedicals, Santa Ana, CA MACSbuffer Miltenyi, San Diego, CA Nonhuman primate CD4 T cell isolation kitMiltenyi, San Diego, CA Propidium iodide (1 mg/mL solution) Sigma, SaintLouis, MO Rhesus monkey (Indian Origin) whole blood, sodium WorldwidePrimates, Miami, FL heparin anti-coagulated RPMI-1640 medium LifeTechnologies, Carlsbad, CA SteadyGlo Luciferase Assay Solution Promega,Madison, WI ViCell counter Beckman Coulter, Indianapolis, IN7.2 Assays7.2.1 Cyno/Rhesus 2-Cell Bioactivity Assays

OX40mAb24 was tested for bioactivity using a 2-cell reporter assay. Ascyno and rhesus monkeys share identical OX40 amino acid sequences, acyno/rhesus OX40-expressing Jurkat NFκB-luciferase reporter cell line(clone B2) was used for readout of OX40 agonism (NFκB activity). Tomediate OX40mAb24 cross-linking and, consequently, OX40 clustering onJurkat reporter cells, either an Fcγ receptor-expressing rhesus B-cellline, LCL8664, or leukocytes from whole blood of a normal rhesus monkey,which contain FcγR expressing cells, were used. Background bioactivitywas assessed both without the addition of OX40mAb24, and also in theabsence of the FcγR-expressing cells. To demonstrate the role of OX40engagement on reporter cells, an isotype control was used in place ofOX40mAb24.

The OX40mAb24 2-cell bioactivity assay with LCL8664 was performedaccording to the following protocol:

The day prior to use, cyno/rhesus OX40-expressing Jurkat NFκB-luciferasereporter clone B2 and LCL8664 were cultured in complete RPMI medium(RPMI with 10% fetal bovine serum [FBS] and 1% antibiotics/antimycotics)to achieve cell densities of approximately 5×10⁶ cells/mL and 4×10⁵cells/mL, respectively. The next day, OX40 Jurkat reporter cells andLCL8664 were pelleted, suspended in complete medium and counted using aViCell counter before cell concentrations for both cell lines wereadjusted to 2.5×10⁶ in complete medium.

Clone B2 and LCL8664 cells were each added to a 96 well round bottom nontissue-culture treated plate at 100,000 cells per well. OX40mAb24 wasadded to cells in complete RPMI medium, to a final concentrationstarting at 30 μg/mL and diluted in 3-fold increments. Similarly, R347IgG1 (isotype control) was used at a final concentration of 10 μg/mL anddiluted in 6-fold increments. 9B12 and MOPC-21 (isotype control) werediluted in the same manner. Plates were transferred to a 37° C.incubator with a humidified 5% CO₂ atmosphere.

After 16 hours incubation time, 100 μL reconstituted Steady-Gloluciferase assay solution was added to each well and mixed to lyse cellsand then incubated to equilibrate luciferase signal. Steady-Glo/samplelysate (150 μL) was transferred from each well to a 96 well, whitewalled assay plate for detection and luminescence read using a PerkinElmer Envision luminescence reader.

The following protocol was used to acquire RBC-lysed rhesus cells fromsodium heparin anti-coagulated whole blood obtained from a healthyIndian-origin rhesus macaque:

Fresh whole blood (5 mL) was added to a 50-mL conical tube and 45 mL ofammonium chloride solution was added. The cell mixture was incubated for10 minutes on ice, then pelleted, and washed with complete RPMI mediumafter which the cells were ready for use in the 2-cell bioactivityassay.

The 2-cell bioactivity assay with rhesus RBC-lysed whole blood cells wasperformed as described with the LCL8664 rhesus B-cell line, but 10 μg/mLNIP228 IgG1 was used as the negative-control antibody.

7.2.2 Rhesus CD4 T Cell Proliferation Assay

OX40mAb24 bioactivity was determined in a CD4⁺ T cell proliferationassay using activated primary rhesus CD4⁺ T cells and plate-captureddrug according to the following protocol. Rhesus CD4⁺ T cells isolatedfrom sodium heparin anti-coagulated whole blood obtained from healthyrhesus monkeys (N=2) from World Wide Primates were used as the source ofresponding cells.

Fresh heparinized rhesus blood was diluted 1:1 with PBS at roomtemperature (RT). Then 20 mL of diluted whole blood was overlayed onto15 mL of 95% lymphocyte separation medium (LSM) in a 50-mL conicalcentrifuge tube. Blood was centrifuged at 400×g for 30 minutes at roomtemperature without the brake. Peripheral blood mononuclear cells werecollected at the interface and washed twice with Miltenyi MACS bufferand pelleted. The cell pellet was treated with red cell lysis buffer for5 minutes, and lysis buffer was deactivated with the addition ofcomplete RPMI medium. The cells were washed and suspended in MACS bufferand counted with a ViCell counter to determine cell number andviability.

Rhesus CD4⁺ T cells were isolated with a Miltenyi nonhuman primate kitaccording to manufacturer's instructions. Then, CD4⁺ T cells werecounted on a ViCell counter, suspended at 1×10⁶ cells/mL in completeRPMI medium with 51 μg/mL Concanavalin A and 1000 IU/mL of IL-2 andcultured at 37° C. and 5% CO₂ in a humidified incubator for 2 days toactivate T cells and induce OX40 expression. Non-tissue culture treatedround-bottom 96 well assay plates were coated with 100 μL of 2 μg/mLgoat anti-mouse Fcγ-specific IgG and 2 μg/mL goat anti-humanFcγ-specific IgG in PBS. Goat anti-human IgG capture antibodies were notadded to wells intended for assay of soluble OX40mAb24 activity. Plateswere incubated overnight at 4° C., washed with 200 μL of PBS, andblocked for 90 minutes at 37° C. with 1% BSA in PBS (1% BSA/PBS). Theplates were washed with PBS and 2 ng/mL of anti-CD3 (clone SP34-2)reconstituted in 1% BSA/PBS was added to the plates for 90 minutes at37° C. The plates were washed with PBS to remove unbound OX40mAb24, R347human IgG1 control mAb, 9B12, and mouse IgG1 control mAb (clone MOPC-21)were each reconstituted in 1% BSA/PBS starting at 1.01 μg/mL (6.67 nM;experiment 1) or 1.5 μg/mL (10.0 nM; experiment 2) and serially dilutedover a 3-fold dilution series and then added to assay plates andincubated for 90 minutes at 37° C.

Activated primary rhesus CD4⁺ T cells were collected, washed in completeRPMI medium, and the concentration adjusted to 1.0×10⁶ viable cells/mL.Cells were labelled with carboxyfluorescein succinimidyl ester (CFSE),according to the manufacturer's instructions, with the exception ofusing 1.25 μM CFSE instead of the recommended 5 μM, with an incubationof 10 minutes at 37° C. After labeling, cells were suspended in completeRPMI and the concentration was adjusted to 1.0×10⁶ per mL. The plateswere washed with PBS and 200 μL of CD4⁺ T cells (200,000/well) was addedto each well. Then, the plate was incubated at 37° C. for 3 days.

After 72 hours incubation time, CD4⁺ T cells were pelleted, and washedonce with PBS containing 2% FBS (FACS buffer). Cells were suspended inbinding mix containing anti-CD4 V450® labeled antibody foridentification of CD4⁺ T cells, and propidium iodide (PI) forlive/non-viable cell discrimination, and incubated for 30 minutes.Following incubation, cells were washed in FACS buffer, re-suspended inFACS buffer and analyzed by flow cytometry using an LSRII flow cytometerand FlowJo software for analysis of Flow Cytometry Standard (FCS)formatted data.

The assay was repeated in two independent experiments using primaryrhesus CD4⁺ T cells.

To assess cell proliferation, live (propidium iodide-negative) eventswere gated using FlowJo software and the percentage of CD4⁺-gated cellsshowing dilution of CFSE was determined. Specific activity of OX40mAb24was calculated by subtracting the percentage of CD4⁺-gated cells showingdilution of CFSE in response to anti-CD3 alone from the percentage ofCD4⁺-gated cells showing dilution of CSFE in response to anti-CD3antibody plus OX40mAb24.

The concentrations of OX40mAb24 that achieved half-maximal (EC₅₀)responses in the Jurkat NFκB reporter assay and the primary rhesus CD4⁺T cell assay were determined using non-linear regression analysis (logdose-response, 4-parameter fit curves) in GraphPad Prism, version 5.01(San Diego, Calif.).

7.3 Results

7.3.1 Cyno/Rhesus 2-Cell Bioactivity Assay

Results of the cyno/rhesus 2-cell bioactivity assays are shown in Table7-2, FIGS. 19A-B, FIGS. 20A-B and FIGS. 21A-B. OX40mAb24 activated theOX40 signaling pathway, as measured by NFκB signaling, in cyno/rhesusOX40-expressing Jurkat T cells in the presence of the rhesus B-cellline, LCL8664, with a mean EC₅₀ of 1450 pM (N=2 experiments). As shownin Table 7-2, the EC₅₀ value of OX40mAb24 activity in the cyno/rhesus2-cell assay was 1.3 fold higher than in human 2-cell assays with theRaji B-cell line, while 9B12 had limited activity in cyno/rhesus 2-cellassays and an EC₅₀ value could not be determined.

In addition, as shown in Table 7-2, OX40mAb24 activated the OX40signaling pathway in

Jurkat T cells in the presence of rhesus RBC-lysed whole blood, which isexpected to contain Fcγ receptor-expressing cells, with an EC₅₀ of 550pM (N=1 experiment). As Table 7-2 also shows, the EC₅₀ value of 9B12 inthe same assay was 6052 pM, and was 4680 pM in a human 2-cell assay withthe Raji B-cell line.

TABLE 7-2 Mean Half Maximal Effective Concentration for OX40mAb24 and9B12 in 2-cell Bioactivity Assays 2-cell assay format Rhesus RBC-lysedRhesus LCL8664 B cells + whole blood + cyno/rhesus OX40- cyno/rhesusOX40- Human Raji B cells + expressing Jurkat NFκB- expressing JurkatNFκB- human OX40 Jurkat luciferase clone B2 luciferase clone B2NFκB-luciferase clone 64 Test or Control bioactivity bioactivitybioactivity^(a) Article [mean EC₅₀ (Std Err); n] [mean EC₅₀ (Std Err);n] [mean EC₅₀ (Std Err); n] OX40mAb24 1450 (204); n = 2  550; n = 1 1110 (92.5); n = 5 R347 human IgG1 no activity; n = 2 NT no activityNIP228 human NT no activity; n = 1 NT IgG1 9B12 ND 6052; n = 1 4680(1220); n = 5 Std Err = standard error of the mean. n = number ofexperiments. NT = Not tested. ND = Not Determined. ^(a)Data from Example5.7.3.2 Rhesus Primary C-cell Proliferation Assay

Results for the rhesus primary T cell proliferation assay are shown inTable 7-3, Table 7-4, and FIGS. 22A-D. OX40mAb24 induced proliferationof primary rhesus CD4⁺ T cells with a mean EC₅₀ of 436 pM (Table 7-3;N=2 experiments). As shown in Table 7-5, the EC₅₀ value for 9B12 was 110pM (Table 7-4; N=2 experiments). In OX40mAb24 induced proliferation ofactivated primary human CD4⁺ T cells in a similar assay format with amean EC₅₀ of 28 pM (Table 7-5; data from Example 4).

TABLE 7-3 Mean Half Maximal Effective Concentration for OX40mAb24 inPrimary Rhesus CD4 T cell Proliferation Assays Test or control No. ofMean EC₅₀ antibody Experiments (Std Err) OX40mAb24 2 436 (309) NIP228IgG1 2 No activity Std Err = standard error of the mean.

TABLE 7-4 Mean Half Maximal Effective Concentration for 9B12 in PrimaryRhesus CD4 T cell Proliferation Assays Test or control No. of Mean EC₅₀antibody Experiments (Std Err) 9B12 2 110 (13) MOPC-21 mouse IgG1 2 Noactivity Std Err = standard error of the mean.

TABLE 7-5 Mean Half Maximal Effective Concentration for OX40mAb24 and9B12 in Rhesus and Human CD4 T cell Proliferation Assays Rhesus MeanHuman Mean Test or control antibody (Std Err) (Std Err) OX40mAb24 436(309)  28 (98) 9B12 110 (13)  218 (35) Std Err = standard error of themean.7.4 Conclusions

OX40mAb24 activated the OX40 signaling pathway in a cyno/rhesus OX40expressing Jurkat T cell NFκB reporter cell line in the presence ofrhesus B cells or Fcγ receptor-expressing cells contained withinRBC-lysed rhesus whole blood. In addition, OX40mAb24 inducedproliferation of primary rhesus CD4⁺ T cells.

Example 8 Activity of OX40mAb24 in Mouse Models of Human Cancers

This example was designed to determine if OX40mAb24 is effective as asingle-agent therapy for the treatment of cancers. This study wasconducted in xenograft models of human cancers mixed with alloreactivehuman T cells in immunocompromised non-obese diabetic/severe combinedimmunodeficient (NOD/SCID) mice.

8.1 Materials

Materials used in this study, and their source, are listed in Table 8-1.

TABLE 8-1 Materials Item Source DMEM medium Invitrogen, Carlsbad, CA FBSInvitrogen, Carlsbad, CA Lymphocyte separation medium VWR, West Chester,PA PBS Invitrogen, Carlsbad, CA RPMI 1640 Invitrogen, Carlsbad, CARosetteSep CD4⁺ T cell Stem Cell Technologies, enrichment kit Vancouver,BC, Canada RosetteSep CD8⁺ T cell Stem Cell Technologies, enrichment kitVancouver, BC, Canada RosetteSep DML medium Stem Cell Technologies,Vancouver, BC, Canada Mitomycin C Sigma-Aldrich, St. Louis, MO8.2 Experimental Protocols8.2.1 Test Animals

Female NOD/SCID mice aged 5 to 9 weeks, were obtained from HarlanLaboratories, Inc. The animals were humanely treated and housedaccording to Iristithtional Animal Care and Use Committee approvedprotocols in the Laboratory Animal Resources facility at MedImmune, anAssociation for Animal Accreditation of Laboratory Animal Care andUnited States Department of Agriculture-licensed facility. The animalswere kept in sterile micro-isolator units, provided with sterile beddingand food, and acidified drinking water ad libitum. Environmentalconditions were standardized (room temperature: 20° C.+/−1° C.; relativehumidity: 50%±10%; 12-hour light-dark cycle). The animals were monitoreddaily for adverse clinical signs and weekly for body weight.

8.2.2 Establishment of Xenografts

Human cancerous A375 cells originating from a human melanoma cell linewere obtained from ATCC. The cells were grown in DMEM with 10% FCS at37° C. under 5% CO₂ in a humidified incubator. They were then harvested,washed once with PBS, then resuspended in PBS.

Human cancerous A375 cells were harvested from cell cultures. They wereresuspended in PBS. A375 cells were subsequently mixed with CD4⁺ andCD8⁺ T cell lines alloreactive to A375 tumor cell lines beforeimplantation into animals.

To generate CD4⁺ and CD8⁺ T cell lines, human peripheral bloodmononuclear cells (PBMCs) from healthy donors were enriched for CD4⁺ orCD8⁺ T cells by the addition of 1 mL RosetteSep T cell enrichmentproduct per 20 mL of whole blood. This was followed by a 20-minuteincubation and subsequent isolation by density gradient centrifugationusing RosetteSep DM-L density medium. After centrifugation, the cellswere washed 3 times with PBS supplemented with 2% fetal bovine serum(FBS) and resuspended in RPMI1640 medium supplemented with 10% FBS.Enriched CD4⁺ and CD8⁺ T cells were cultured separately for 7 to 10 daysin medium supplemented with recombinant human interleukin 2 (rhIL-2) andeach combined with mitomycin C-treated A375 cells. T cells werecollected and separately cultured again for 7 to 10 days in mediumsupplemented with rhIL-2 and combined with mitomycin C-treated A375cells. CD4⁺ and CD8⁺ T cells were collected and combined at a 2:1 ratio.

A375 cells and PMBCs enriched for CD4⁺ and CD8⁺ T cells were mixed at aratio of 6 A375 cells to 1 T cell immediately before implantation.

Xenografts were established by subcutaneous (SC) injection of 3.5×10⁶cells (human T cells mixed with A375 cells at an effector-to-target(E:T) ratio of 1:6 and suspended in 200 μL of PBS) into the right flanksof the animals.

8.2.3 Randomization, Group Designation, and Dose Levels

Six animals were randomly assigned to each experimental group prior toSC injection of cells. There were no animal substitutions. Groupdesignations and dose levels for each experiment are listed in Table8-2, Table 8-3 and Table 8-4. Test and control antibodies were dilutedin PBS to appropriate concentrations and administered intraperitoneally(IP) in a total volume of 200 μL. The first dose of test and controlantibodies was administered on Day 3 or 4 after implantation ofcancer/effector T cells. The animals received up to 3 additional dosesof the test and control antibodies, as indicated in the figures anddescribed in the corresponding figure description. The formation oftumors was observed in each animal 1 or 2 times a week. The primaryendpoints in this study was either a tumor volume of 2000 mm³ or grosstumor necrosis.

8.2.4 Tumor Measurements

Tumors were measured at intervals indicated in the figures and tablesfor each experiment by caliper and tumor volumes (V) were calculatedusing the following formula:V(mm³)=(length [mm]×width [mm]×width [mm])/2.

-   -   For each group, the results are reported as the arithmetic mean.        Antitumor effects were expressed as percent tumor growth        inhibition (% TGI), which was calculated as follows:        % TGI=(1−[mean tumor V of treatment group]÷[mean tumor V of        control group])×100        8.3 Statistical Methods

A comparison between OX40mAb24-treated or 9B12-treated, and isotypecontrol-treated animals was made, and intergroup differences wereanalyzed for statistical significance by a Mann-Whitney rank sum test.

Significant p-values obtained from the Mann-Whitney rank sum test arepresented in the summary tables and figures adjacent to the arithmeticmean and standard deviation of the mean.

8.4 Results

The activity of OX40mAb24 on growth of tumors in mouse models of humancancer was investigated in this study. Immunodeficient NOD/SCID femaleanimals were implanted with human cancer cell lines mixed withalloreactive human CD4⁺ and CD8⁺ T cell lines. CD4⁺ and CD8⁺ T cellswere derived from PBMCs isolated from healthy human donors. Animalsreceived the first dose of the test and control antibodies there or fourdays after implantation of xenographs, and were administered additionaldoses of the test and control antibodies as indicated.

In three separate experiments, OX40mAb24 plus alloreactive human T cellssignificantly inhibited growth of A375 cells by up to 85% as compared tothe isotype-control group (Table 8-2, Table 8-3, and Table 8-4; FIG.23A, FIG. 24A and FIG. 25). The control 9B12 plus alloreactive human Tcells also significantly inhibited growth of A375 cells by up to 77% ascompared to the isotype-control group (Tables 8-2 to 8-4, FIG. 23B andFIG. 24B.

TABLE 8-2 Treatment Groups and Percent TGI in A375 Xenograft Model onDay 18 (Experiment 2) Dose ^(b) % Group ^(a) Test Antibody (mg/kg) TGI^(c) 1 None; no T cells NA NA 2 None NA NA 3 Isotype control 5 NA 4OX40mAb24 5 79 5 OX40mAb24 2.5 75 6 OX40mAb24 1.0 85 7 9B12 5 53 IP =intraperitoneal; NA = not applicable; TGI = tumor growth inhibition; V =volume ^(a) Number of animals per group: 6. ^(b) All animals received200 μl of test antibody IP on Days 4, 7, 9, and 12 ^(c) % TGI = [1 −(mean tumor V of treatment group) ÷ (mean tumor V of isotype controlgroup)] × 100

TABLE 8-3 Treatment Groups and Percent TGI in A375 Xenograft Model onDay 25 (Experiment 1) Dose ^(b) % Group ^(a) Test Antibody (mg/kg) TGI^(c) 1 None; no T cells NA NA 2 None NA NA 3 Isotype control 5 NA 4OX40mAb24 5 68 5 OX40mAb24 2.5 83 6 OX40mAb24 1.0 84 7 9B12 5 80 IP =intraperitoneal; NA = not applicable; TGI = tumor growth inhibition; V =volume ^(a) Number of animals per group: 6. ^(b) All animals received200 μl of test antibody IP on Days 4, 7, 9, and 12 ^(c) % TGI = [1 −(mean tumor V of treatment group) ÷ (mean tumor V of isotype controlgroup)] × 100

TABLE 8-4 Treatment Groups and Percent TGI in A375 Xenograft Model onDay 28 (Experiment 3) Dose ^(b) % Group ^(a) Test Antibody (mg/kg) TGI^(c) 1 None; no T cells NA NA 2 None NA NA 3 Isotype control 3.0 NA 4OX40mAb24 3.0 75 5 OX40mAb24 1.0 73 6 OX40mAb24 0.3 68 7 OX40mAb24 0.184 8 OX40mAb24 0.03 73 IP = intraperitoneal; NA = not applicable; TGI =tumor growth inhibition; V = volume ^(a) n = 6. ^(b) All animalsreceived 200 μl of test antibody IP on Days 3, 6, 8, and 10 ^(c) % TGI =[1 − (mean tumor V of treatment group) ÷ (mean tumor V of isotypecontrol group)] × 1008.5 Conclusions

OX40mAb24 demonstrated potent antitumor activity in mouse models ofhuman cancer mixed with alloreactive human T cells. The antitumoractivity of OX40mAb24 was similar to the antitumor activity of 9B12.These results provide evidence that OX40mAb24 can be effective as asingle-agent therapy for the treatment of patients with cancer with a Tcell infiltrate.

Example 9 Rat/Mouse Anti-Mouse OX40 IgG2a Chimera Antibody Clone OX86Inhibits the Growth of Mouse Cancer Cell Lines in Syngeneic Models

OX40mAb24 does not cross-react to mouse (m)OX40 (See Example 2);therefore, it is not possible to test its activity in immunocompetentmouse models. OX86 is a rat anti-mOX40 IgG1 antibody that specificallybinds to and triggers signaling of mOX40 (al-Shamkhani et al., Eur. J.Immunol. 26:1695-1699 (1996)), and has anti-tumor activity inimmunocompetent mouse models of cancer (Weinberg A D, et al., J.Immunol. 164:2160-2169 (2000)). To more fully study the effects of OX40agonism using a mouse surrogate antibody with functional propertiessimilar to OX40mAb24, a rat/mouse anti-mOX40 IgG2a chimera antibody(OX86 mIgG2a; rat anti-OX40 light and heavy chain variable regions withmouse IgG2a constant regions) was generated from OX86. This exampleevaluates the single-agent anti-tumor activity of OX86 mIgG2a in threemouse models of cancer.

9.1 Materials

Materials used in this study, and their source, are listed in Table 9-1.

TABLE 9-1 Materials Item Source Phosphate-buffered saline, pH 7.2 LifeTechnologies, Carlsbad, CA Fetal bovine serum, heat inactivated LifeTechnologies, Carlsbad, CA Roswell Park Memorial Institute LifeTechnologies, Carlsbad, CA 1640 medium 0.25% Trypsin-EDTA (1x) LifeTechnologies, Carlsbad, CA EDTA = Ethylenediaminetetraacetic acid.9.2 Experimental Protocols9.2.1 Test Animals

BALB/c and C57BL/6 mice of 6-8 weeks of age were received at MedImmunefrom Harlan Laboratories, Inc. (Indianapolis, Ind.) and allowed toacclimatize for 3 days prior to study start. Thereafter, mice wereshaved at tumor implantation sites and implanted with microchiptransponders for identification.

The animals were housed according to Institutional Animal Care and UseCommittee approved protocols in the Laboratory Animal Resources facilityat MedImmune, an Association for Animal Accreditation of LaboratoryAnimal Care and United States Department of Agriculture-licensedfacility. The animals were kept in sterile micro-isolator units,provided with sterile bedding and food, and acidified drinking water adlibitum. Environmental conditions were standardized (room temperature:20° C.±1° C.; relative humidity: 50%±10%; 12-hour light-dark cycle). Theanimals were monitored daily for adverse clinical signs and bi-weeklyfor body weight. If hind limb paralysis, respiratory distress, 20% bodyweight loss, or tumor volume greater than 2000 mm³ were noted, theanimals were immediately sacrificed humanely by asphyxiation with CO₂.

9.2.2 Establishment and Implantation of Syngeneic Tumors

CT26 and 4T1 were obtained from ATCC in Manassas, Va. MCA205 cells wereobtrained from Providence Cancer Center, Portland, Oreg. All cells werecultured in RPMI 1640 cell culture medium supplemented with 10% FBS andgrown at 37° C. at 5% CO₂ in a humidified tissue culture chamber, thenharvested, washed once in FBS and then resuspended in PBS.

Allografts were established by subcutaneous (SC) injection of 5.0×10⁵CT26 cells, or 1.0×10⁵ 4 T1 cells suspended in 0.1 mL of PBS into theright flank of 7- to 9-week-old BALB/c mice, while 2.5×10⁵ MCA205 cellssuspended in 0.1 mL of phosphate-buffered saline were injected into theright flank of 7- to 9-week-old C57BL/6 mice.

9.2.3 Randomization, Group Designation, and Dose Levels

BALB/c (total of 216) and C57BL/6 (total of 70) female mice were used inthis study. BALB/c mice implanted with CT26 tumor cells were randomlyassigned after tumors grew to a mean volume of 120 mm³ per cohort, 9days after implantation or 200 mm³ per cohort, 13 days afterimplantation. BALB/c mice implanted with 4T1 tumor cells were randomlyassigned after tumors grew to a mean volume of 120 mm³ per cohort, 13days after implantation. C57BL/6 mice were randomly assigned aftertumors grew to a meanvolume of 95 mm³ per cohort, 11 days afterimplantation. Group designations number of animals, dose levels, anddose schedule are presented in Table 9-2, Table 9-3, Table 9-4, andTable 9-5. All test antibodies and control antibodies were administeredby intraperitoneal (IP) injection. There were no animal substitutions.

Animals from each group were sacrificed when tumor volumes reachedapproximately 2000 mm³ or when tumors became ulcerated or necrotic.

TABLE 9-2 Study Design: CT26 Syngeneic Model Number of Dose animalsschedule Dose level Group (M/F) Treatment (study day) (mg/kg)^(a) ROA 110 (F) None NA NA IP 2 10 (F) Negative control 9, 12 2.5 IP (OX86 mIgG1D265A) 3 10 (F) OX86 mIgG2a 9, 12 2.5 IP 4 10 (F) OX86 mIgG2a 9, 12 1.0IP 5 10 (F) OX86 mIgG2a 9, 12 0.25 IP 6 10 (F) OX86 mIgG2a 9, 12 0.1 IPF = female; IgG1 = immunoglobulin G1; IP = intraperitoneal; mAb =monoclonal antibody; OX86 mIgG2a = rat anti-OX40 light and heavy chainvariable regions of clone OX86 with mouse IgG2a constant regions; OX86mIgG1 D265A = mouse anti-mouse OX40 mIgG1 with a point mutation in theFc domain that reduces its ability to bind Fcγ receptors; NA = notapplicable because the animals were not treated; ROA = route ofadministration. ^(a)Dose volume: 0.2 mL.

TABLE 9-3 Study Design: CT26 Syngeneic Model Number of Dose animalsschedule Dose level Group (M/F) Treatment (study day) (mg/kg)^(a) ROA 112 (F) None NA NA IP 2 12 (F) OX86 mIgG2a 13, 16 10 IP 3 12 (F) OX86mIgG2a 13, 16 3 IP 4 12 (F) OX86 mIgG2a 13, 16 1 IP 5 12 (F) OX86 mIgG2a13, 16 0.3 IP 6 12 (F) OX86 mIgG2a 13, 16 0.1 IP F = female; IP =intraperitoneal; 0X86 mIgG2a = rat anti-OX40 light and heavy chainvariable regions of clone OX86 with mouse IgG2a constant regions; NA =not applicable because the animals were not treated; ROA = route ofadministration. ^(a)Dose volume: 0.2 mL.

TABLE 9-4 Study Design: MCA205 Syngeneic Model Number of Dose animalsschedule Dose level Group (M/F) Treatment (study day) (mg/kg)^(a) ROA 114 (F) None NA NA IP 2 14 (F) Isotype control 11, 14 20 IP (mixture)^(b)3 14 (F) OX86 mIgG2a 11, 14 10 IP 4 14 (F) OX86 mIgG2a 11, 14 10 IP 5 14(F) OX86 mIgG2a 11, 14  5 IP F = female; IP = intraperitoneal; 0X86mIgG2a = rat anti-OX40 light and heavy chain variable regions of cloneOX86 with mouse IgG2a constant regions; NA = not applicable because theanimals were not treated; ROA = route of administration. ^(a)Dosevolume: 0.2 mL. ^(b)Mixture contains isotype control antibodies with Fcdomains of mIgG2a (10 mg/kg) and mIgG2b (10 mg/kg).

TABLE 9-5 Study Design: 4T1 Syngeneic Model Number of Dose animalsschedule Dose level Group (M/F) Treatment (study day) (mg/kg)^(a) ROA 112 (F) None NA NA IP 2 12 (F) Isotype control 13, 16, 20, 23 70 IP(mixture)^(b) 3 12 (F) OX86 mIgG2a 13, 16, 20, 23 10 IP 4 12 (F) OX86mIgG2a 13, 16, 20, 23 5 IP 5 12 (F) OX86 mIgG2a 13, 16, 20, 23 2.5 IP 612 (F) OX86 mIgG2a 13, 16, 20, 23 1.0 IP 7 12 (F) OX86 mIgG2a 13, 16,20, 23 0.25 IP F = female; IP = intraperitoneal; 0X86 mIgG2a = ratanti-OX40 light and heavy chain variable regions of clone OX86 withmouse IgG2a constant regions; NA = not applicable because the animalswere not treated; ROA = route of administration. ^(a)Dose volume: 0.2mL. ^(b)Mixture contains isotype control antibodies with Fc domains ofmIgG2a (10 mg/kg), mIgG2b (20 mg/kg), rat IgG2a (20 mg/kg) and rat IgG2b(20 mg/kg).9.2.4 Tumor Measurements

Tumors were measured by caliper twice weekly and tumor volumes werecalculated using the following formula:tumor volume (V)=[length (mm)×width (mm)²]/2

-   -   where length was defined as the larger side and width as the        smaller side perpendicular to the length.

Antitumor effects of each group were expressed as tumor growthinhibition (TGI), which was calculated as follows:% TGI=(1−[mean V of treatment group]÷[mean V of control group]×100

-   -   Tumor growth responses were categorized as a complete response        (CR) if there was no measureable tumor.        9.3 Statistical Methods

One-way ANOVA was used to determine mean tumor volume differences. Inthe event of a significant F test a Dunnett's or Sidak's multiplecomparison test was utilized (where appropriate). Where applicable, alog 10 transformation was applied to tumor volumes to account forheteroscedasticity. A p value<0.05 was considered significant.

9.4 Results

Treatment of mice with the OX86 mIgG2a results in significantly reducedgrowth of CT26 and MCA205 tumor cells compared to untreated or negative,or isotype control antibody-treated mice control, (Table 9-6, Table 9-7and Table 9-8; FIG. 26A, FIG. 27A, and FIG. 28A). Treatment of mice withthe OX86 mIgG2a results in delayed and reduced growth of 4T1 tumor cellscompared to untreated or isotype control antibody (Table 9-9 and FIG.29A).

A mixed response is often shown in syngeneic models; however, thedramatic response of the treatment with OX86 mIgG2a is clearer from theindividual animal tumor growth graphs (FIG. 26B, FIG. 27B, FIG. 28B andFIG. 29B). A dose response was not observed in mice treated with OX86mIgG2a based on TGI (Table 9-6, Table 9-7, Table 9-8 and Table 9-9; FIG.26A, FIG. 27A, FIG. 28A and FIG. 29A) or with respect to increasing thenumber of animals exhibiting complete responses (Table 9-6, Table 9-7,Table 9-8 and Table 9-9).

TABLE 9-6 Treatment Groups, Percent Tumor Growth Inhibition on Day 22,and number of Complete Responders in CT26 Syngeneic Model Number ofComplete Test/Control Dose ^(b) % Responders out of Group ^(a) Antibody(mg/kg) TGI ^(c) 12 mice ^(d) 1 None NA NA 0 2 OX86 mIgG2a 10 67 8 3OX86 mIgG2a 3 67 6 4 OX86 mIgG2a 10 75 8 5 OX86 mIgG2a 0.3 65 8 6 OX86mIgG2a 0.1 70 8 IP = intraperitoneal; NA = not applicable; TGI = tumorgrowth inhibition; V volume. ^(a) n = 12. ^(b) All animals received 200μL of test antibody IP on Days 13 and 16. ^(c) % TGI = [1 − (mean tumorV of treatment group) ÷ (mean tumor V of control group)] × 100. ^(d)Number of animals in a group with a tumor volume measurement recorded aszero at the end of the study.

TABLE 9-7 Treatment Groups, Percent Tumor Growth Inhibition on Day 26,and number of Complete Responders in CT26 Syngeneic Model Number ofComplete Test/Control Dose ^(b) % Responders out of Group ^(a) Antibody(mg/kg) TGI ^(c) 10 mice ^(d) 1 None NA NA 1 2 Negative control 2.5 NA 1(OX86 mIgG1 D265A) 3 OX86 mIgG2a 2.5 94 7 4 OX86 mIgG2a 1.0 97 8 5 OX86mIgG2a 0.25 95 6 6 OX86 mIgG2a 0.1 92 7 IP = intraperitoneal; NA = notapplicable; TGI = tumor growth inhibition; V volume. ^(a) n = 10. ^(b)All animals received 200 μL of test antibody IP on Days 9 and 12. ^(c) %TGI = [1 − (mean tumor V of treatment group) ÷ (mean tumor V of controlgroup)] × 100. ^(d) Number of animals in a group with a tumor volumemeasurement recorded as zero at the end of the study.

TABLE 9-8 Treatment Groups, Percent Tumor Growth Inhibition on Day 22,and Number of Complete Responders in MCA205 Syngeneic Model Number ofComplete Test/Control Dose ^(b) % Responders out of Group ^(a) Antibody(mg/kg) TGI ^(c) 14 mice ^(d) 1 None NA NA 0 2 Isotype control 20 NA 0(mixture) 3 OX86 mIgG2a 20 65 0 4 OX86 mIgG2a 10 76 4 5 OX86 mIgG2a  570 2

TABLE 9-9 Treatment Groups, Percent Tumor Growth Inhibition on Day 25,and number of Complete Responders in 4T1 Syngeneic Model Number ofComplete Test/Control Dose ^(b) % Responders out of Group ^(a) Antibody(mg/kg) TGI ^(c) 12 mice ^(d) 1 None NA NA 0 2 Isotype control 70 NA 0(mixture) 3 OX86 mIgG2a 10  5 0 4 OX86 mIgG2a 5 34 2 5 OX86 mIgG2a 2.537 0 6 OX86 mIgG2a 1.0 22 0 7 OX86 mIgG2a 0.25 ND 0 IP =intraperitoneal; NA = not applicable; TGI = tumor growth inhibition; Vvolume; ND = not determined. ^(a) n = 12. ^(b) All animals received 200μL of test antibody IP on Days 13, 16, 20 and 23. ^(c) % TGI = [1 −(mean tumor V of treatment group) ÷ (mean tumor V of control group)] ×100. ^(d) Number of animals in a group with a tumor volume measurementrecorded as zero at the end of the study.9-5 Conclusions

Single-agent treatment of tumor-bearing mice with OX86 mIgG2a results inanti-tumor activity that significantly reduces growth of multiple tumorsas compared to untreated, negative control, and/or isotype controltreated groups.

Example 10 Characterization of the Epitope of OX40mAb24

This example examines the epitope of OX40mAb24 using a series ofhuman/mouse OX40 chimeric variants.

10.1 Materials Used in this Study, and their Source, are Listed in Table10-1.

TABLE 10-1 Materials Item Source Anti-human IgG-Alexa480 Invitrogen(Carlsbad, CA) Anti-sheep IgG-Alexa-480 Invitrogen (Carlsbad, CA)Anti-goat IgG-Alexa480 Invitrogen (Carlsbad, CA) FreeStyle 293F cellsInvitrogen (Carlsbad, CA) (HEK293F cells) Goat anti-mouse OX40polyclonal R&D Systems (Minneapolis, MN) antibody LSRII flow cytometerBD Biosciences (San Jose, CA) Phosphate buffered saline Invitrogen(Carlsbad, CA) Sheep anti-human OX40 polyclonal R&D Systems(Minneapolis, MN) antibody 293fectin transfection reagent Invitrogen(Carlsbad, CA)10.2 Generation of Human/Mouse Chimeric Variants

OX40mAb24 binds specifically to human OX40 (SEQ ID NO: 91) and does notrecognize mouse OX40 (SEQ ID NO: 92), despite sharing 60% identity intheir amino acid (aa) sequence (FIG. 30). Human/mouse chimeric OX40variants were engineered by swapping portions of the extracellulardomain between human and mouse OX40. The cDNA constructs of human andmouse OX40 were used as templates in overlapping extension PCR toconstruct chimeric variants with a transmembrane domain for cell-surfaceexpression of the recombinant proteins. OX40 is an approximately 45 kDaprotein with three complete cysteine-rich domains (CRDs) and onetruncated cysteine-rich domain. The structure is characteristic of theTNFR superfamily.

Thirteen chimeric knock-out (KO/loss-of-function) variants wereconstructed by replacing the following residues of the extracellulardomain of human OX40 with the corresponding mouse OX40 aa or Alanine(FIG. 31):

-   -   CRD1 (human OX40 aa 29 to 65 replaced with the mouse        counterparts);    -   CRD2 (human OX40 aa 66 to 107 replaced with the mouse        counterparts);    -   CRD3 (human OX40 aa 108 to 146 replaced with the mouse        counterparts);    -   CRD4+linker (human OX40 aa 147 to 214 replaced with the mouse        counterparts);    -   CRD3+4 (human OX40 aa 108 to 167 replaced with its mouse        counterparts);    -   A¹¹¹ (human OX40 aa alanine 111 replaced with its mouse        counterpart proline);    -   L¹¹⁶ (human OX40 aa leucine 116 replaced with its mouse        counterpart glutamine);    -   P¹²¹ (human OX40 aa proline 121 replaced with its mouse        counterpart leucine);    -   A¹²⁶ (human OX40 aa alanine 126 replaced with its mouse        counterpart valine);    -   D¹³⁷ (human OX40 aa aspartic acid 137 replaced with its mouse        counterpart asparagine);    -   A¹¹¹P¹²¹D¹³⁷ (human OX40 aa Ala111, Pro121 and Asp137 replaced        with the mouse counterparts);    -   L¹¹⁶A¹²⁶ (human OX40 aa Leu116 and Ala126 replaced with the        mouse counterparts); and    -   D¹¹⁷S¹¹⁸ (human OX40 aa Asp117 and Ser118 replaced with Ala        mutations).

In addition, five knock-in (KI/gain-of-function) variants wereconstructed by grafting the following residues of human OX40 into mouseOX40 using the same method as described above for the KO constructs(FIG. 31):

-   -   CRD3 (human OX40 aa 108 to 146 grafted into the corresponding        mouse regions);    -   A¹¹¹ (human OX40 aa alanine 111 grafted into the corresponding        mouse position);    -   L¹¹⁶ (human OX40 aa leucine 116 grafted into the corresponding        mouse position);    -   A¹²⁶ (human OX40 aa alanine 126 grafted into the corresponding        mouse position);    -   L¹¹⁶A¹²⁶ (human OX40 aa leucine 116 and alanine 126 grafted into        the corresponding mouse positions).

The resulting chimeric DNAs were closed into a mammalian expressionvector pEBNA for transient mammalian expression. 293F cells were seededat a density of 0.7×10⁶ cells/mL one day prior to transfection. Threeand a half micro grams of each expression vector were transfected into 5mL of HEK293 F cells using 5 μL of 293fectin transfection reagentfollowing the manufacturer's instructions.

10.3 Characterization of Binding of OX40mAb24 to Chimeric OX40 Variants

Forty-eight hours after transfection, HEK293F cells were incubated with1 μg/mL of OX40mAb24 for 1 hour on ice in PBS. To detect bound OX40mAb24by flow cytometry, the cells were washed 3-times with cold PBS,incubated with 1 μg/mL of Alexa480-conjugated anti-human IgG antibodyfor 1 hr on ice, and then analyzed using the LSRII flow cytometer.

The expression levels of all chimeric OX40 variants were also monitoredby flow cytometry; the cells were incubated with a mixture of sheepanti-human OX40 and goat anti-mouse OX40 polyclonal antibodies at 1μg/mL in PBS for 1 hour on ice. Cells were washed 3-times with cold PBS,and then incubated with a mixture of Alexa480-conjugated anti-goat andanti-sheep IgG polyclonal antibodies. After washing 3 times with coldPBS, cells were analyzed with the LSRII flow cytometer.

10.4 Results

The epitope of OX40mAb24 was characterized using chimeric human/mousevariants. Human OX40 and mouse OX40 share 60% identity in the aasequence (FIG. 30); yet, OX40mAb24 binds specifically to human OX40 anddoes not recognize mouse OX40. This specificity was employed to identifythe epitope of OX40mAb24. Eighteen chimeric variants were constructed byswapping in or out various domains of the extracellular domain of mouseOX40 into human OX40 (KO/loss-of-function variants) or of human OX40into mouse OX40 (KI/gain-of-function variants) (FIG. 31). All variantsencoded a transmembrane domain for cell-surface expression of thechimeric protein. The binding characteristics of OX40mAb24 to thesevariants were analyzed by flow cytometry.

The epitope of OX40mAb24 was mapped to the CRD3 domain of human OX40 byswapping individual domains between human and mouse OX40s. The bindingof OX40mAb24 to human OX40 was abolished when replacing human CRD3domain with the mouse counterpart (KO_CRD3 and KO_CRD3-4) (FIGS. 32A-C).Replacement of the other human CRD domains with mouse regions did notaffect the binding of OX40mAb24(KO_CRD1, KO_CRD2, and KO_CRD4+linker).Furthermore, grafting the human CRD3 domain into mouse OX40 led to thebinding of OX40mAb24 (KI_CRD3). All variants were expressed as monitoredby anti-human and mouse OX40s polyclonal antibodies (FIGS. 32A-C). Theparental mAb of OX40mAb24, 9B12, was also characterized in the bindingstudy. 9B12 shows the same binding profiles to these variants asOX40mAb24, suggesting both IgGs recognize the same epitope of the CRD3domain.

Furthermore, critical epitope residues Leu¹¹⁶ and Ala¹²⁶ were identifiedin the CRD3 domain by mutating the amino acids that are differentbetween human and mouse OX40s. Five amino acids, including Ala¹¹¹,Leu¹¹⁶, Pro¹²¹, Ala¹²⁶, and Asp¹³⁷ (FIG. 30) in the human CRD3 domain,were mutated to corresponding mouse residues or Ala as individual aminoacids or different combinations (FIG. 31). The binding of OX40mAb24 wasabolished when replacing human residues Leu¹¹⁶ and Ala¹²⁶ with the mousecounterparts (KO_L¹¹⁶+A¹²⁶). Furthermore, the KI/gain-of-functionvariants confirmed the importance of these two critical residues.Grafting Leu¹¹⁶, Ala¹²⁶, or the combination into mouse OX40 led to thebinding of OX40mAb24.

10.5 Conclusions

The epitope OX40mAb24 was mapped to the CRD3 domain of human OX40 usinghuman/mouse chimeric variants, with critical residues Leu¹¹⁶ and Ala¹²⁶.The binding profiles to all variants between OX40mAb24 and its parental9B12 are identical, indicating they recognize the same epitope on humanOX40.

Example 11 Pharmacodynamic Activity of the Rat/Mouse Anti-Mouse OX40IgG2a Chimera Antibody Clone OX86 in Naïve Mice and in Mice BearingSyngeneic Tumors

OX86 is a rat anti-mOX40 IgG1 antibody that specifically binds to andtriggers signaling of MOX40 (al-Shamkhani et al, 1996), and hasanti-tumor activity in immunocompetent mouse models of cancer (Weinberget al, 2000). To more fully study the effects of OX40 agonism using amouse surrogate antibody with functional properties similar toOX40mAb24, this example utilizes the rat/mouse anti-mOX40 IgG2a chimeraantibody OX86 described in Example 9. However, it is possible to drawsome parallels between the species, for example mIgG2a and human IgG1are often considered equivalent functionally as both isotypes are ableto bind multiple Fcγ receptors, are capable of high affinity Fcγreceptor binding and are able to trigger ADCC and bind to human C1q, aprerequisite for complement-dependent cytotoxicity (CDC) by theclassical complement pathway (Stewart et al. 2014; Dall'Acqua et al,2006). OX86 mIGg2a binds to mouse OX40 (see Example 9) and was used as asurrogate mouse OX40 agonist antibody for OX40mAb24.

In this example, the ability of OX86 mIgG2a to induce T cells in naïveand tumor bearing mice, and to enter the cell cycle and proliferate.Further, K167 and ICOS were evaluated as a potential biomarker of OX40agonist activity, was investigated. T-cell proliferation and anti-tumoractivity was determined in two syngeneic mouse models of cancer.Moreover, finally, the role of activating (Fcγ receptors I, III and IV)and/or inhibitory (Cfy receptor IIb) Fcγ receptors play in vivo activityof OX86 mIgG2a was determined.

11.1 Materials and Methods

11.1.1 Animal Receipt and Identification

Wild-type Fcgr2b^(−/−) or Fcer1g^(−/−) BALB/c and Fcgr2b^(−/−) orFcer1g^(−/−) C57BL/6 mice of 6-8 weeks of age were received at MedImmunefrom Harlan Laboratories, Inc. (Indianapolis, Ind. or Charles RiverLaboratories (UK)) and allowed to acclimatize for <15 days prior tostudy start. Thereafter, mice were implanted with microchip transpondersto identify individual mice.

11.1.2 Housing

The animals were humanely treated and housed according to InstitutionalAnimal Care and Use Committee (USA) and Home Office (UK) approvedprotocols in the Laboratory Animal Resources facility (USA) andBiological Sciences Unit (UK) at MedImmune, an Association for AnimalAccreditation of Laboratory Animal Care and United States Department ofAgriculture-licensed facility. The animals were kept in sterilemicro-isolator units, provided with sterile bedding and food, andacidified drinking water (USA) or tap water (UK) ad libitum.Environmental conditions were standardized (room temperature: 20° C.±1°C. (USA) or 21° C.±1° C. (UK); relative humidity: 50%±10% (USA) or55±10% (UK); 12-hour light-dark cycle). The animals were monitored dailyfor adverse clinical signs and bi-weekly for body weight. If hind limbparalysis, respiratory distress, 20% body weight loss, or tumor volumegreater than 2000 mm³ were noted, the animals were immediatelysacrificed humanely by cervical dislocation or asphyxiation with CO₂.

11.1.3 Establishment of Syngeneic Tumors

The CT26 cell line (mouse colon carcinoma) was obtained from ATCC,Manassas, Va., and the cell line MCA 205 (chemically-induced mouse softtissue sarcoma) was obtained from the Providence Cancer Center,Portland, Oreg. Both cell lines were maintained in RPMI 1640 medium+10%FBS at 37° C., 5% CO₂.

Allografts were established by subcutaneous (SC) injection of 5.0×10⁵CT26 cells, resuspended in 0.1 mL of PBS, into the right flank of 7- to9-week-old wild-type, Fcgr2b^(−/−) or Fcer1g^(−/−) BALB/c mice.Allografts were also established by SC injection of 2.5×10⁵ MCA205cells, resuspended in 0.1 mL of PBS, into the right flank of 7- to9-week-old wild-type, Fcgr2b^(−/−) or Fcer1g^(−/−) C57BL/6 mice.

11.1.4 Randomization, Group Designation and Dose Levels

Wild-type (n=70), Fcgr2b^(−/−) (n=36) or Fcer1g^(−/−) (n=36) BALB/c andFcgr2b^(−/−) (n=36) or Fcer1g^(−/−)C57BL/6 female mice (n=36) were usedin this study. All mice were randomly assigned into treatment groups.Group designations, number of animals, dose levels and dose schedule arepresented in Table 11-1, Table 11-2 and Table 11-3. All test articlesand control articles were administered intraperitoneally (IP). Therewere no animal substitutions.

Animals from each group were sacrificed when tumor volumes reachedapproximately 2000 mm³ or when tumors became ulcerated or necrotic.

TABLE 11-1 Group Designation and Dose Levels of Naïve Balb/c Mice Numberof Dose animals schedule Dose level Group (M/F) Treatment (study day)(mg/kg)^(a) ROA 1, 9  7 (F), 7 (F) Saline 1 NA IP 3, 11 7 (F), 7 (F)Isotype control 1 20 IP (NIP228 IgG2a) 4, 12 7 (F) OX86 mIgG2a 1 20 IP5, 13 7 (F), 7 (F) OX86 mIgG2a 1 2 IP 6, 14 7 (F), 7 (F) OX86 mIgG2a 10.2 IP F = female; M = male; NA = not applicable; IP = intraperitoneal;ROA = route of administration; OX86 mIgG2a = mouse anti-mouse OX40 IgG2amonoclonal antibody variant of OX86 ^(a)Dose volume: 0.2 mL for salineor volume adjusted to body weight; 10 mL/kg for all other groups.

TABLE 11-2 Group Designation and Dose Levels in the CT26 Syngeneic ModelNumber of Dose Dose animals Mouse schedule level Group (M/F) StrainTreatment (study day) (mg/kg)^(a) ROA 1 8 (F) Balb/c None NA NA IP 2 8(F) Fcgr2b^(−/−) Control 4, 7 2.5 IP (OX86 mIgG1 D265A) 3 8 (F) OX86mIgG2a 4, 7 2.5 IP 4 8 (F) Balb/c None NA NA IP 5 8 (F) Fcer1g^(−/−)Control 4, 7 2.5 IP (OX86 mIgG1 D265A) 6 8 (F) OX86 mIgG2a 4, 7 2.5 IP F= female; M = male; NA = not applicable because the animals were nottreated; IP = intraperitoneal; ROA = route of administration; OX86mIgG2a = rat anti-OX40 light and heavy chain variable regions of cloneOX86 with mouse IgG2a constant regions. ^(a)Dose volume: 0.2 mL.

TABLE 11-3 Group Designation and Dose Levels in the MCA205 SyngeneicModel Number of Dose Dose animals Mouse schedule level Group (M/F)Strain Treatment (study day) (mg/kg)^(a) ROA 1 8 (F) C57BL/6 None NA NAIP 2 8 (F) Fcgr2b^(−/−) Control 4, 7 7.5 IP (OX86 mIgG1 D265A) 3 8 (F)OX86 mIgG2a 4, 7 7.5 IP 4 8 (F) C57BL/6 None NA NA IP 5 8 (F)Fcer1g^(−/−) Control 4, 7 7.5 IP (OX86 mIgG1 D265A) 6 8 (F) OX86 mIgG2a4, 7 7.5 IP F = female; M = male; NA = not applicable because theanimals were not treated; IP = intraperitoneal; ROA = route ofadministration; OX86 mIgG2a = rat anti-OX40 light and heavy chainvariable regions of clone OX86 with mouse IgG2a constant regions.^(a)Dose volume: 0.2 mL.11.1.5 Tumor Measurements

Tumors were measured by caliper twice weekly and tumor volumes werecalculated using the following formula:tumor volume=[length(mm)×width(mm)²]/2

-   -   where length was defined as the larger side, and width as the        smaller side perpendicular to the length.

Antitumor effects of each group were expressed as tumor growthinhibition (TGI), which was calculated as follows:% TGI=(1−[mean tumor V of treatment group]+[mean tumor V of controlgroup])×10011.1.6 Tissue Collection and Single Cell Isolation11.1.6.1 Preparation of Mouse Blood Cells for Flow Cytometry Analysis

Red blood cell lysis (RBCL) buffer (2 mL) was added to blood (50 μL) andincubated for 5 min. Volumes obtained from in-life bleeds ranged from 20μL to 50 μL. Terminal bleeds were 50 μL. RPMI+10% fetal bovine serum(FBS) (8 mL) was added to each sample. Cells in each sample werepelleted (300×g, 5 min) and then resuspended in 0.3 mL Flow Buffer. Cellsuspensions (200 μL) were added to each well of a 96-well round-bottomedplate for staining with fluorescent antibodies. Pooled group sampleswere used for unstained, the single antibody staining, isotype stainingand the fluorescence-minus-one (FMO) antibody staining controls.

11.1.6.2 Mouse Draining Lymph Node and Spleen Isolation and Generationof Single Cell Suspensions for Flow Cytometry Analysis

Spleens were placed in 10 mL of RPMI+1× Pen/Strep solution and passedthrough a 40 μm cell strainer. Samples were pelleted (300×g, 5 min) andthen resuspended in 1 mL RBCL buffer for 3 min. RPMI+FBS 10% (9 mL) wasadded to each sample. Cell suspensions were pelleted (300×g, 5 min) andresuspended in 1 mL of Flow Buffer. Cell suspensions (100 μL) were addedto each well of a 96-well round-bottomed plate for staining withfluorescent antibodies. Pooled group samples were used for unstained,the single antibody staining, isotype staining and FMO antibody stainingcontrols.

11.1.6.3 Preparation of Single-Cell Suspension of Mouse Tumor for FlowCytometry Analysis

Tumors were aseptically excised from euthanized mice, being careful toavoid collecting connective tissue or skin, and placed in collagenasediluted with Hanks balanced salt solution in a 6-well dish. To increasethe efficiency of the enzymatic digestion process, each tumor was mincedwith a scalpel or a small pair of sharp scissors into smaller pieces.Minced tumors were transferred into 15 mL conical tubes and placed on ashaking platform to incubate at 37° C. for 20-30 minutes. Five mL ofRPMI+10% FBS was added to each sample to deactivate the collagenase andmaintain viability of the immune cells. Samples were passed through a 70μM cell strainer and placed into 50 mL conical tubes. An aliquot of eachsample was removed for counting. The remaining amount sample waspelleted in a centrifuge at 330×g and resuspended in FACS buffer (PBS+2%FBS) at a concentration of 1×10⁷ cells per mL.

11.1.7 Flow Cytometry Analysis

11.1.7.1 Analysis of Tissues from Non-tumor-bearing Mice

Single-cell suspensions of blood or spleen cells were pelleted (300×g, 5min), resuspended in 50 μL of Fc Block solution (1:50), diluted ineBiosciences Flow Buffer and incubated for 10 min on ice. Fiftymicroliters of fluorescently labelled antibodies (2× stock solution)were added to each sample (final volume 100 μL). Single antibodystaining solutions (extracellular) were added at 1 μL per well andcell/antibody mixtures were incubated for 30 min on ice. Cells werepelleted (300×g, 5 min), washed twice (200 μL Flow Buffer per well,300×g, 5 min), resuspended in 50 μL Fix/Penn buffer (one partconcentrate to three part diluent) and then incubated overnight at 4° C.in the dark.

Treated cells were washed twice in 1× Permeabilization Buffer (dilutedin water). Intracellular labelling antibodies were diluted intoPermeabilization Buffer (100 μL per well) and added to the cells, whichwere then incubated at room temperature in the dark for 30 minutes.Antibodies for single marker staining (intracellular) were added tocells at 1 μL per well into 100 jut Permeabilization Buffer andincubated for 30 min on ice in the dark. Cells were washed once inPermeabilization Buffer and resuspended in 3.7% formaldehyde solution(100 μL) before being analyzed on a Canto II flow cytometer (BDBiosciences, San Jose, Calif.).

11.1.7.2 Analysis of Tissues from Tumor-bearing Mice

11.1.7.2.1 Cell Surface Staining

Each sample of pelleted cells was resuspended in FACS Buffer.Fluorescent antibody mixtures in FACS Buffer were added to appropriatewells and incubated for 20-30 minutes at 4° C. in the dark. Cells werewashed twice with FACS Buffer, resuspended in FACS Buffer and analyzedon a LSRII flow cytometer (BD Biosciences, San Jose, Calif.).

11.1.7.2.2 Intracellular Staining (Fixed and Permeabilized Cells)

Each sample of pelleted cells was resuspended in 50 μL of a 1:500dilution of a fluorescence fixable blue dye for live/non-viable celldiscrimination, and incubated for 15 minutes at 4° C. in the dark. Cellswere washed once with FACS Buffer, resuspended in 30 μL PBS+4% normalmouse serum containing Fc Block, and incubated for 15 minutes at roomtemperature. Fluorescent antibody mixtures in FACS Buffer were added toappropriate wells, and incubated for 20-30 minutes at 4° C. in the dark.Cells were washed twice with FACS Buffer, resuspended in 150 μL freshlyprepared FoxP3 Fix/Penn working solution and incubated at roomtemperature for 30 minutes in the dark. Cells were washed twice with 1×Permeabilization Buffer, resuspended in 100 μL 1× PermeabilizationBuffer containing antibodies to stain intracellular antigens, andincubated at room temperature for 30 minutes in the dark. Cells werewashed once with 1× Permeabilization Buffer, resuspended in FACS Bufferand analyzed on a LSRII flow cytometer.

11.1.8 Statistical Methods

One-way ANOVA was used to determine mean tumor volume differences andmean percentage of Ki67+ or ICOS+ cells. In the event of a significant Ftest, a Dunnett's or Sidak's multiple comparison test was utilized(where appropriate). Where applicable, a log 10 transformation wasapplied to mean values to account for heteroscedasticity. A p value<0.05was considered significant. GraphPad Prism 6.0 was employed for linearregression analysis which generated the best fit line that bestpredicted Y from X, the level of statistical significance of the lineand the goodness of fit (r²) of the determined best fit line.

11.1.9 Materials

Materials used in this study, and their source, are listed in Table 11-4and Table 11-5.

TABLE 11-4 Materials Item Source 0.25% Trypsin-EDTA (1X) LifeTechnologies, Carlsbad, CA 10 x Permeabilization Buffer eBioscience, UK3.7% Formaldehyde solution Sigma-Aldrich, UK 40 and 70 μm cell strainersCorning Life Sciences, Tewksbury, MA Collagenase type 3 WorthingtonBiochem Corp., Lakewood, NJ Fc Block eBioscience, UK Fetal bovine serum,heat inactivated Life Technologies, Carlsbad, CAFixation/Permeabilization Concentrate eBioscience, UKFixation/Permeabilization Diluent eBioscience, UK Flow BuffereBioscience, UK FoxP3 Fix/Perm Kit eBioscience, UK Hanks buffered saltsolution Life Technologies USA Heat Inactivated Gamma Irradiated FBS forSASC Biosciences, KS, USA RBCL Buffer neutralization LSRII and Canto IIFlow Cytometers BD Biosciences, San Jose, CA Normal mouse serum JacksonLabs, Bar harbor, MA Pen/Strep solution Life Technologies UKPermeabilization Buffer eBiosciences, USA Phosphate buffered saline, pH7.2 Life Technologies, Carlsbad, CA Red Blood Cell Lysis Buffer Sigma,UK Roswell Park Memorial Institute 1640 medium Life Technologies,Carlsbad, CA

TABLE 11-5 Fluorescent Antibodies for Flow Cytometry Studies Item SourceAPC conjugated Rat IgG2a anti-CD4 eBiosciences, UK FITC conjugatedmIgG2a anti-MHC2 eBiosciences USA APC-H7 conjugated Rat IgG2a anti-CD8BD Biosciences, UK EFluor 480 conjugated Rat IgG2a anti-Ki67eBioscience, UK APC conjugated isotype control Rat IgG2a eBioscience, UKAPC-H7 conjugated isotype control Rat IgGa BD Biosciences, UK eFluorconjugated isotype control Rat IgG2a eBioscience, UK PE conjugatedisotype control Rat IgG2b eBioscience, UK PECy7 conjugated rlgG2a Ki67eBioscience, USA BV605 conjugated rlgG2a anti CD4 Biolegend USA BV711conjugated rlgG2a anti CD8 Biolegend USA Fixable Blue Life Technologies,USA PeCy7 conjugated Isotype control rlgG2a Biolegend USA11.2 Results

Intraperitoneal treatment of naïve mice with the anti-OX40 antibody OX86mouse IgG2a (OX86 mIgG2a) resulted in a significant, dose-dependent andtransient increase in the percentage of Ki67+CD4+ and ICOS+CD4+ T cellsin the blood over time, compared to mice treated with control articles(FIGS. 33A and C). The largest percentage of Ki67+CD4+ and ICOS+CD4+ Tcells was detected on Day 10. A significant increase in the percentageof Ki67+CD4+ and ICOS+CD4+ T cells was measured in the spleens of miceon Day 10 following the administration of OX86 mIgG2a, compared to micetreated with control articles (FIGS. 33B and D). A statisticallysignificant and strong correlation between the percentage of Ki67+CD4+ Tcells and ICOS+CD4+ T cells in the blood and spleen on Day 10 wasidentified (FIGS. 34A and 34B).

Treatment of naïve mice with the OX86 mIgG2a also resulted in asignificant, transient increase in the percentage of Ki67+CD8+ T cellsin the blood over time, when compared to mice treated with controlarticles (FIG. 35A). Increases in the percentage of ICOS+CD8+ T cells inthe blood over time (FIG. 35C), and the percentage of Ki67+CD8+ T cellsin the spleen on Day 10 (FIG. 35B), were detected following treatmentwith OX86 mIgG2a, but were not statistically significant when comparedto treatment with control articles. Treatment of naïve mice with OX86mIgG2a did induce a significant, dose-dependent increase in thepercentage of ICOS+CD8+ T cells in the spleen on Day 10, compared tomice treated with control articles (FIG. 35D). Moderate but significantcorrelations between the percentage of Ki67+CD8+ T cells and ICOS+CD8+ Tcells in the blood and spleen was determined (FIGS. 36A and 36B).

Single-agent treatment of tumor-bearing wild-type mice with OX86 mIgG2aresults in an antitumor activity that reduces growth of twohistologically different tumors as compared to untreated and isotypecontrol treated groups (See Example 9). Treatment with OX86 mIGg2aresulted in significantly reduced growth of MCA205 tumors on Day 20(FIG. 38A, Table 11-7) when compared to treatment with control articlesin C57BL/6 mice that were genetically engineered to lack expression ofthe inhibitory Fcγ receptor IIB (Fcgr2b^(−/−) mice). Identical treatmentof mice with CT26 tumors resulted in a reduced growth of tumors that didnot reach statistical significance on Day 18 (FIG. 37A, Table 11-6) whencompared to treatment with control articles in Fcgr2b^(−/−)BALB/c mice.No inhibition of growth, by any agent, was observed in tumor bearingBalb/c and C57BL/6 mice genetically engineered to lack expression of theactivating Fcγ receptors (Fcer1g^(−/−) mice; FIG. 37C; FIG. 38C). Mixedresponse is often observed in syngeneic models. However, the anti-tumorresponse following treatment with OX86 mIgG2a in the two differentstrains of Fcγ receptor knock-out mice can also be discerned from theindividual animal tumor growth graphs (FIGS. 37B and D; FIGS. 38B andD); treatment with OX86 mIgG2a resulted in the inhibition of CT26 andMCA205 tumor growth and in some cases induced complete responses inFcgr2b^(−/−) mice (FIG. 37B; FIG. 38B).

TABLE 11-6 Treatment Groups and Percent TGI and Number of CompleteResponders in CT26 Syngeneic Mouse Model Number of Complete Test/ControlDose ^(b) Mouse % Responders out of Group ^(a) Article (mg/kg) StrainTGI ^(c) 8 mice ^(d) 1 None NA Balb/c NA 0 2 Isotype control 2.5 mg/kgFcgr2b^(−/−) NA 0 3 OX86 mIgG2a 2.5 mg/kg 15 0 4 None NA Balb/c NA 0 5Isotype control 2.5 mg/kg Fcer1g^(−/−) NA 0 6 OX86 mIgG2a 2.5 mg/kg <0 0TGI = tumor growth inhibition; NA = not applicable; IP =intraperitoneal; V = volume ^(a) Number of animals per group: 8. ^(b)All animals received 200 μL of test article IP on Days 4 and 7. ^(c) %TGI = [1 − (mean tumor V of treatment group) ÷ (mean tumor V of controlgroup)] × 100; calculated on Day 18 for Fcgr2b^(−/−) mice and Day 16 forFcer1g^(−/−) mice ^(d) Number of animals in a group with a tumor volumemeasurement recorded as zero at the end of the study.

TABLE 11-7 Treatment Groups and Percent TGI on Day 20 and Number ofComplete Responders in MCA205 Syngeneic Mouse Model Number of CompleteTest/Control Dose ^(b) Mouse % Responders out of Group ^(a) Article(mg/kg) Strain TGI ^(c) 8 mice ^(d) 1 None NA C57BL/6 NA 0 2 Isotypecontrol 7.5 mg/kg Fcgr2b^(−/−) NA 0 3 OX86 mIgG2a 7.5 mg/kg 95 8 4 NoneNA C57BL/6 NA 0 5 Isotype control 7.5 mg/kg Fcer1g^(−/−) NA 0 6 OX86mIgG2a 7.5 mg/kg 35 1 TGI = tumor growth inhibition; NA = notapplicable; IP = intraperitoneal; V = volume ^(a) Number of animals pergroup: 8. ^(b) All animals received 200 μL of test article IP on Days 4and 7. ^(c) % TGI = [1 − (mean tumor V of treatment group) ÷ (mean tumorV of control group)] × 100 ^(d) Number of animals in a group with atumor volume measurement recorded as zero at the end of the study.

In parallel pharmacodynamics studies, treatment with OX86 mIgG2acompared to control articles caused a significant increase in thepercentage of Ki67+CD4+ T cells in the spleen (FIG. 39A) and Ki67+CD8+ Tcells in the peripheral blood and the spleen, but not in the tumor (FIG.40A), of CT26 tumor bearing Fcgr2b^(−/−)Balb/c mice. No increase inKi67+CD4+ or CD8+ T cells was detected in the blood, spleen or tumor ofCT26 tumor bearing Fcer1g^(−/−)Balb/c mice following treatment with OX86mIgG2a, as compared to control articles (FIG. 39B; FIG. 40B).

Additionally, in parallel studies, treatment with OX86 mIgG2a comparedto control articles resulted in a significant increase in the percentageof Ki67+CD4+ T cells in the draining lymph node, spleen and tumor (FIG.41A) and Ki67+CD8+ T cells in the draining lymph node and the spleen(FIG. 42A) of MCA205 tumor bearing Fcgr2b^(−/−) C57BL/6 mice. Increasesin the percentage of Ki67+CD8+ T cells in the tumor of MCA205 tumorbearing Fcgr2b^(−/−)C57BL/6 mice were not statistically significant(FIG. 42A). Treatment with OX86 mIgG2a as compared to control articlesalso induced a significant increase in the percentage of Ki67+CD4+ Tcells in the draining lymph node and spleen of MCA205 tumor bearingFcer1g^(−/−)C57BL/6 mice (FIG. 41B). A significant increase in thepercentage of Ki67+CD8+ T cells in the spleen of these mice was alsoobserved (FIG. 42B). No significant increases in the percentage ofKi67+CD4+ T cells in the tumor (FIG. 42B) or Ki67+CD8+ T cells in thedraining lymph node or tumor of MCA205 tumor bearing Fcgr2b^(−/−)C57BL/6mice were detected (FIG. 42B).

11.3 Conclusions

A single dose of OX86 mIgG2a in naïve mice induced a transient increasein T-cell activation and proliferation as measured by increasedexpression of ICOS and Ki67, respectively. A significant linearcorrelation of the percentage of ICOS and Ki67 on CD4+ and CD8+ T cellswas found. Antitumor activity of OX86 mIgG2a, measured as inhibition ofthe growth of CT26 and MCA205 tumors, was dependent on the expression ofactivating Fcγ receptors I, III and IV, but not the inhibitory Fcγreceptor IIB. Increases in peripheral blood, draining lymph node and/orspleen T-cell proliferation (Ki67) were observed in parallelpharmacodynamic experiments in tumor-bearing mice that expressed theactivating Fcγ receptors; some T-cell proliferation was observed in micethat expressed only the inhibitory Fcγ receptor. Together with thepharmacodynamic results from naïve mice and while not wishing to bebound by theory, a potential mechanism for OX86 mIgG2a antitumoractivity is increased peripheral and intratumoral T-cell proliferation(Ki67). Moreover, OX86 mIgG2a in vivo antitumor activity occurs uponexpression of activating Fcγ receptors.

Example 12 T Cell Pharmacodynamic Changes in Response to OX40mAb24Therapy in Immunocompromised Mice Engrafted with Human HematopoieticStem Cells

This example investigates whether OX40mAb24 can activate and expandhuman CD4+ and CD8+ memory T cells or reduce human Tregs inimmunocompromised mice with a reconstituted human immune system.OX40mAb24 was tested in an in vivo mouse model system reconstituted withhuman immune cells to determine if the drug has immunomodulatory effectson human T cells.

12.1 Materials and Methods

12.1.1 Test Animals

Nod.Cg-Prkdc^(scid)Il2rg^(tm1Wy)/SzJ (NSG) Mice (n=14 ages 23-26 weeks)were obtained from the Jackson Laboratory. The animals were humanelytreated and housed according to Institutional Animal Care and UseCommittee approved protocols in the Laboratory Animal Resources facilityat MedImmune, an Association for Animal Accreditation of LaboratoryAnimal Care and United States Department of Agriculture-licensedfacility. The animals were kept in sterile micro-isolator units,provided with sterile bedding and food, and acidified drinking water adlibitum. Environmental conditions were standardized (room temperature:20° C.±1° C.; relative humidity: 50%±10%; 12-hour light-dark cycle). Theanimals were monitored daily for adverse clinical signs during thecourse of the study.

12.1.2 Pharmacodynamics of OX40mAb24 in HSC Engrafted (Humanized) Mice

12.1.2.1 Establishment of Human HSC Engrafted Mice

Human CD34+HSC engrafted mice were purchased from the JacksonLaboratory. Mice were generated as follows: pooled human CD34+ cellsfrom multiple umbilical cord blood donors were isolated and injectedintravenously into the tail vein of Nod.Cg-Prkdc^(scid)112re^(tm1Wy)/SzJ (NSG) mice. At 12 weeks after engraftment, mouseperipheral blood was sampled and lysed with Pharm Lyse red blood celllysis buffer and then analyzed by flow cytometry for human CD45, CD19and CD3 positive cells to determine the degree of human immune cellengraftment in mice. Mice successfully engrafted with 25% or more CD45+human immune cells of total viable blood cells were subsequently shippedto MedImmune.

12.1.2.2 Antigen Challenge and OX40mAb24 Treatment of Human HSCEngrafted Mice

The sustained engraftment of human immune cells into NSG mice wasconfirmed by flow cytometry at 22 weeks post injection of human CD34cells, and at 23 weeks mice were placed into 3 groups that were leftuntouched or received treatment (Table 12-1). There were nostatistically significant differences in the mean percentage of humanCD45+ cell engraftment between eventual treatment groups at week 22prior to the start of the experiment. Group 1 consisted of 4 mice anddid not receive subcutaneous (SC) keyhole limpet hemocyanin (KLH)immunization or antibody administration. Group 2 consisted of 5 mice andreceived 300 μg/50 μL of KLH and 50 μL of complete Freund's adjuvant(CFA) at two independent injection SC sites. This group of mice alsoreceived 2 mg/kg of hIgG1 isotype control antibody, administeredintraperitoneally (IP), one day after KLH immunization. Group 3consisted of 5 mice and received KLH/CFA SC immunization as describedabove. In addition, mice were also administered one day later a singleIP dose of 2 mg/kg OX40mAb24. On the day of KLH/CFA SC immunization,prior to immunization (pre-treatment), and seven days later(post-treatment), whole blood was obtained through retro-orbital bleedand cells were immunophenotyped and counted by flow cytometry.

TABLE 12-1 Experimental Groups, Human CD45+ Cell Engraftment, andTreatment Human CD45+ Cells as a Percentage of Total Viable Human PlusMouse CD45 Cells^(a) - Treatment^(b) - Mouse Week 22 post Week 23 postGroup Number HSC engraftment HSC engraftment 1 1-1 4575 39.5 None 1 1-24576 68.3 None 1 1-3 4577 20.9 None 1 1-4 4578 23.8 None 2 2-1 4579 29.6KLH immunization + NIP228 huIgG1 2 2-2 4580 21.7 KLH immunization +NIP228 huIgG1 2 2-3 4581 27.8 KLH immunization + NIP228 huIgG1 2 2-44582 50.3 KLH immunization + NIP228 huIgG1 2 2-5 4583 32.5 KLHimmunization + NIP228 huIgG1 3 3-1 4584 15.4 KLH immunization +OX40mAb24 3 3-2 4585 44.3 KLH immunization + OX40mAb24 3 3-3 4586 20.3KLH immunization + OX40mAb24 3 3-4 4587 16.3 KLH immunization +OX40mAb24 3 3-5 4588 25.6 KLH immunization + OX40mAb24 Hu = human; KLH =keyhole limpet hemocyanin; HSC = hematopoietic stem cells. ^(a)HumanCD45+ cells determined by flow cytometry; ^(b)KLH immunization, keyholelimpet hemocyanin plus complete Freund's adjuvant.12.1.2.3 Immune Cell Analysis by Flow Cytometry

Cells were immunophenotyped by flow cytometry using a whole bloodantibody binding protocol. Briefly, EDTA anti-coagulated whole blood ata constant volume was added to wells of a deep well plate and a T cellstaining master mix added to cells to bind cell surface antigens. Afterwashes in FACS buffer (PBS, pH 7.2 plus 2% heat-inactivated newborn calfserum), red blood cells (RBC) were lysed using 1×RBC lysis buffer, andcells fixed and permeabilized using EBiosciences Fix and Perm kitaccording to the manufacturer's protocol. Subsequently, anti-FoxP3 andanti-Ki67 mAbs were bound to cells. Cells were washed using 1× fix andperm buffer, re-suspended in FACS buffer and events analyzed using anLSRII flow cytometer. Raw flow cytometry standard (FCS) data wasanalyzed using FlowJo software, and cell populations identified afterlive/dead cell discrimination and removal of doublets and cell debris.After gating for CD4+ and CD8+ T cell populations, memory T cells weredetermined by CD45RA and CCR7 expression profile, with naïve T cellsdefined as CD45RA+/CCR7+, effector T cells (Teff) as CD45RA+/CCR7−,effector memory T cells (Tem) as CD4SRA−/CCR7−, and central memory Tcells (Tcm) as CD45RA−/CCR7+. Tregs were defined as CD4+/FoxP3+ cells.

12.1.2.4 Statistical Methods

GraphPad Prism software for Windows (version 6.03) was used for graphingand statistical analysis of data.

One-way ANOVA multiple comparisons test with Turkey's post-test analysiswas used to compare differences between experimental group means. In theevent of a significant F test, a Sidak's multiple comparisons test wasutilized. A p value of less than 0.05 was considered significant.

12.1.3 Materials

Materials used in this study, and their source, are listed in Table12-2.

TABLE 12-2 Materials Item Source Anti-human CCR7 APC clone 150503 R&DSystems, Minneapolis, MN Anti-human CD14 APC clone M5E2 Biolegend, SanDiego, CA Anti-human CD16 PE clone 1243 Biolegend, San Diego, CAAnti-human CD25 FITC clone M-A251 Biolegend, San Diego, CA Anti-humanCD3 FITC clone UCHT1 Biolegend, San Diego, CA Anti-human CD4 BV605 cloneRPA-T4 Becton Dickinson, San Jose, CA Anti-human CD56 FITC clone HCD56Biolegend, San Diego, CA Anti-human CD8 PE-CF594 clone RPA-T8 BectonDickinson, San Jose, CA Anti-human CD19 APC clone HIB19 Biolegend, SanDiego, CA Anti-human CD19 PE-CF594 clone HIB19 Becton Dickinson, SanJose, CA Anti-human CD45 PE clone HI30 Biolegend, San Diego, CAAnti-human CD45 PE clone HI30 Biolegend, San Diego, CA Anti-human FoxP3PE clone PHC101 eBioscience, San Diego, CA Anti-human HLA-DR BV421 clone3G8 Biolegend, San Diego, CA Anti-human Ki67 FITC clone B56 BectonDickinson, San Jose, CA Anti-mouse CD16/32 PE-Cy7 Biolegend, San Diego,CA Anti-mouse CD45 PE-Cy7 clone 30F11 Biolegend, San Diego, CA CompleteFreund's adjuvant (CFA) Difco ™ Becton Dickinson, San Jose, CAEBiosciences Fix and Perm Kit EBioscience, San Diego, CA FlowJo SoftwareFlowJo, Ashland, OR FoxP3/Transcription Factor eBioscience, San Diego,CA Fixation/Permeabilization Concentrate and Diluent Graphpad Prismsoftware, v 6.03 Graphpad Software, San Diego, CA Heat-inactivatednewborn calf serum Life Technologies, Frederick, MD Keyhole limpethemocyanin Thermo Fisher Scientific, Waltham, MA Live/Dead ® FixableBlue Dye Life Technologies, Frederick, MD LSR II flow cytometer BDBiosciences, San Jose, CA Mouse IgG2a APC isotype control eBioscience,San Diego, CA Mouse IgG2b BV421 isotype control Biolegend, San Diego, CAMouse IgG1 FITC isotype control Biolegend, San Diego, CAPhosphate-buffered saline (PBS), pH 7.2 Life Technologies, Frederick, MDRat IgG1 PE-CF594 isotype control Becton Dickinson, San Jose, CA RatIgG2a PE isotype control eBioscience, San Diego, CA Rat IgG2a PE-Cy7isotype control Biolegend, San Diego, CA12.2 Results

OX40mAb24 or huIgG1 control antibody was administered one day afterimmunization with KLH. OX40mAb24 resulted in a statistically significantreduction in the percentage of Treg in peripheral blood after six daysrelative to the group administered a huIgG1 isotype control antibody(p=0.019, one-way ANOVA; FIGS. 43A and 43B). No differences in thepercentages of Tregs between treatment groups were observed prior toimmunization and antibody administration.

In addition to a significant decrease in the percentage of peripheralblood Tregs, there was a statistically significant increase in the CD4+Tem:Treg ratio following treatment with OX40mAb24 relative to huIgG1isotype matched control (p=0.042), and trends towards significantincreases for total CD4+:Treg (p=0.051), CD4+ Teff:Treg (p=0.10), andCD4+ Tem:Treg (p=0.069) ratios (FIGS. 44A-D) Likewise, a trend towardssignificance was observed for an increase in the CD8+ Teff:Treg ratio(p=0.064) in peripheral blood of mice treated with OX40mAb24 relative tothat of mice treated with huIgG1 isotype control (FIGS. 45A-B).

CD25 (IL-2 receptor) is up-regulated on T cells following activation,and is considered a marker of recently activated T cells. Increases inthe percentage of CD25 positive CD8+ T cells were observed for theOX40mAb24 treatment group relative to the huIgG1 treatment group fortotal CD8+ (p=0.038) effector CD8+ (p=0.076), and for effector memoryCD8+ (p=0.040) T cells (FIGS. 46A-C). Therefore, agonism of OX40 byOX40mAb24 activated CD8+ T cells relative to the control antibody.

12.3 Conclusions

In mice engrafted with human immune cells, treatment with OX40mAb24after antigen immunization resulted in decreased peripheral Treg cellsrelative to mice receiving a human IgG1 control antibody. Likewise, theratio of total, effector, and memory CD4+ T cells, and effector CD8+ Tcells, relative to Treg cells were also increased in the peripheralblood of mice receiving OX40mAb24 compared with those receiving huIgG1control antibody. Finally, the percentage of CD8+ T cells expressingCD25 on the cell surface was increased after OX40mAb24 treatment,indicating an OX40 induction of peripheral CD8+ T-cell activation.

TABLE OF SEQUENCES SEQ ID NO Description Sequence  1 9B12 VLDIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSKLHSGVPSRFSGSGSRTDYSLTITDLDQEDIATYFCQQGSALPWTFGQGTKVEIK  2 LCDR1 RASQDISNYLN  3 LCDR2YTSKLHS  4 LCDR3 QQGSALPWT  5 9B12 VHEVQLQESGPSLVKPSQTLSLTCSVTGDSFTSGYWNWIRKFPGNRLEYMGYISYNGITYHNPSLKSRISITRDTSKNHYYLQLNSVTTEDTATYFCARYRYDYDGGHAMDYWGQGTLVTVSS  6 HFW1QVQLQESGPGLVKPSQTLSLTCAVYGGSFS  7 HFW1-variantQVQLQESGPGLVKPSQTLSLTCAVYGDSFS  8 HCDR1 SGYWN  9 HFW2-XXXWIRX₃₉HPGKGLEX₄₇X₄₈G; where X₃₉ is Q or K, X₄₇ is W or Y, and X₄₈ is Ior M 10 HFW2-variant WIRQHPGKGLEWIG 11 HFW2-variant WIRKHPGKGLEYMG 12HFW2-variant WIRKHPGKGLEWIG 13 HFW2-variant WIRKHPGKGLEYIG 14 HCDR2YISYNGITYHNPSLKS 15 HCDR2-variant YISYNAITYHNPSLKS 16 HCDR2-variantYISYSGITYHNPSLKS 17 HFW3-XXXRITINX₇₁DTSKNQX₇₈SLQLNSVTPEDTAVYX₉₁CAR;, where X₇₁ is P or R, X₇₈ is For Y, and X₉₁ is Y or F 18 HFW3-variant RITINPDTSKNQFSLQLNSVTPEDTAVYYCAR19 HFW3-variant RITINRDTSKNQYSLQLNSVTPEDTAVYFCAR 20 HFW3-variantRITINRDTSKNQFSLQLNSVTPEDTAVYYCAR 21 HFW3-variantRITINRDTSKNQFSLQLNSVTPEDTAVYFCAR 22 HFW3-variantRITINRDTSKNQYSLQLNSVTPEDTAVYYCAR 23 HFW3-variantRITINPDTSKNQYSLQLNSVTPEDTAVYFCAR 24 HFW3-variantRITINPDTSKNQYSLQLNSVTPEDTAVYYCAR 25 HCDR3 YRYDYDGGHAMDY 26 HCDR3-variantYKYDYDAGHAMDY 27 HCDR3-variant YKYDYDGGHAMDY 28 HFW4 WGQGTLVTVSS 29OX40mAb VLDIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSKLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGSALPWTFGQGTKVEIK 30 OX40mAb lightDIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSKLHSGVPSRFSGSGSchainGTDYTLTISSLQPEDFATYYCQQGSALPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 31 OX40Mab lightGACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACAGAGTGACCAT chain DNACACCTGTCGGGCCAGCCAGGACATCAGCAACTACCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACTACACCAGCAAGCTGCACAGCGGCGTGCCCAGCAGATTCAGCGGCAGCGGCTCCGGCACCGACTACACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGGCTCCGCCCTGCCCTGGACCTTTGGCCAGGGCACCAAGGTGGAAATCAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT 32 OX40mAb VL-hu2DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAVKLLIYYTSKLHSGVPSRFSGSGSRTDYTLTISSLQPEDFATYYCQQGSALPWTFGQGTKVEIK 33 OX40mAb5 VHQVQLQESGPGLVKPSQTLSLTCAVYGGSFSSGYWNWIRQHPGKGLEWIGYISYNGITYHNPSLKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARYRYDYDGGHAMDYWGQGTLVTVSS 34 OX40mAb5 VHCAGGTGCAGCTGCAGGAAAGCGGCCCTGGCCTGGTCAAGCCCAGCCAGACCCTGAGCCTGAC DNACTGTGCCGTGTACGGCGGCAGCTTCAGCAGCGGCTACTGGAACTGGATCCGGCAGCACCCCGGCAAGGGCCTGGAATGGATCGGCTACATCAGCTACAACGGCATCACCTACCACAACCCCAGCCTGAAGTCCCGGATCACCATCAACCCCGACACCAGCAAGAACCAGTTCTCCCTGCAGCTGAACAGCGTGACCCCCGAGGACACCGCCGTGTACTACTGCGCCCGGTACAGATACGACTACGACGGCGGCCACGCCATGGACTACTGGGGCCAGGGCACCCTGGTCACCGTGTCCTCT 35 OX40mAb8 VHQVQLQESGPGLVKPSQTLSLTCAVYGGSFSSGYWNWIRKHPGKGLEWIGYISYNGITYHNPSLKSRITINRDTSKNQFSLQLNSVTPEDTAVYYCARYRYDYDGGHAMDYWGQGTLVTVSS 36 OX40mAb8VH CAGGTGCAGCTGCAGGAAAGCGGCCCTGGCCTGGTCAAGCCCAGCCAGACCCTGAGCCTGAC DNACTGTGCCGTGTACGGCGGCAGCTTCAGCAGCGGCTACTGGAACTGGATCCGGAAGCACCCCGGCAAGGGCCTGGAATGGATCGGCTACATCAGCTACAACGGCATCACCTACCACAACCCCAGCCTGAAGTCCCGGATCACCATCAACCGGGACACCAGCAAGAACCAGTTCTCCCTGCAGCTGAACAGCGTGACCCCCGAGGACACCGCCGTGTACTACTGCGCCCGGTACAGATACGACTACGACGGCGGCCACGCCATGGACTACTGGGGCCAGGGCACCCTGGTCACCGTGTCCTCT 37 OX40mAb13 VHQVQLQESGPGLVKPSQTLSLTCAVYGDSFSSGYWNWIRKHPGKGLEYMGYISYNGITYHNPSLKSRITINRDTSKNQYSLQLNSVTPEDTAVYFCARYRYDYDGGHAMDYWGQGTLVTVSS 38 OX40mAb13 VHCAGGTGCAGCTGCAGGAAAGCGGCCCTGGCCTGGTCAAGCCCAGCCAGACCCTGAGCCTGAC DNACTGTGCCGTGTACGGCGACAGCTTCAGCAGCGGCTACTGGAACTGGATCCGGAAGCACCCCGGCAAGGGCCTGGAATACATGGGCTACATCAGCTACAACGGCATCACCTACCACAACCCCAGCCTGAAGTCCCGGATCACCATCAACCGGGACACCAGCAAGAACCAGTACTCCCTGCAGCTGAACAGCGTGACCCCCGAGGACACCGCCGTGTACTTCTGCGCCCGGTACAGATACGACTACGACGGCGGCCACGCCATGGACTACTGGGGCCAGGGCACCCTGGTCACCGTGTCCTCT 39 OX40mAb14 VHQVQLQESGPGLVKPSQTLSLTCAVYGDSFSSGYWNWIRKHPGKGLEYIGYISYNGITYHNPSLKSRITINRDTSKNQYSLQLNSVTPEDTAVYFCARYRYDYDGGHAMDYWGQGTLVTVSS 40 OX40mAb14 VHCAGGTGCAGCTGCAGGAAAGCGGCCCTGGCCTGGTCAAGCCCAGCCAGACCCTGAGCCTGAC DNACTGTGCCGTGTACGGCGACAGCTTCAGCAGCGGCTACTGGAACTGGATCCGGAAGCACCCCGGCAAGGGCCTGGAATACATCGGCTACATCAGCTACAACGGCATCACCTACCACAACCCCAGCCTGAAGTCCCGGATCACCATCAACCGGGACACCAGCAAGAACCAGTACTCCCTGCAGCTGAACAGCGTGACCCCCGAGGACACCGCCGTGTACTTCTGCGCCCGGTACAGATACGACTACGACGGCGGCCACGCCATGGACTACTGGGGCCAGGGCACCCTGGTCACCGTGTCCTCT 41 OX40mAb15 VHQVQLQESGPGLVKPSQTLSLTCAVYGDSFSSGYWNWIRKHPGKGLEYIGYISYNGITYHNPSLKSRITINRDTSKNQFSLQLNSVTPEDTAVYFCARYRYDYDGGHAMDYWGQGTLVTVSS 42 OX40mAb15 VHCAGGTGCAGCTGCAGGAAAGCGGCCCTGGCCTGGTCAAGCCCAGCCAGACCCTGAGCCTGAC DNACTGTGCCGTGTACGGCGACAGCTTCAGCAGCGGCTACTGGAACTGGATCCGGAAGCACCCCGGCAAGGGCCTGGAATACATCGGCTACATCAGCTACAACGGCATCACCTACCACAACCCCAGCCTGAAGTCCCGGATCACCATCAACCGGGACACCAGCAAGAACCAGTTCTCCCTGCAGCTGAACAGCGTGACCCCCGAGGACACCGCCGTGTACTTCTGCGCCCGGTACAGATACGACTACGACGGCGGCCACGCCATGGACTACTGGGGCCAGGGCACCCTGGTCACCGTGTCCTCT 43 OX40mAb16 VHQVQLQESGPGLVKPSQTLSLTCAVYGDSFSSGYWNWIRKHPGKGLEYIGYISYNGITYHNPSLKSRITINRDTSKNQYSLQLNSVTPEDTAVYYCARYRYDYDGGHAMDYWGQGTLVTVSS 44 OX40mAb16 VHCAGGTGCAGCTGCAGGAAAGCGGCCCTGGCCTGGTCAAGCCCAGCCAGACCCTGAGCCTGAC DNACTGTGCCGTGTACGGCGACAGCTTCAGCAGCGGCTACTGGAACTGGATCCGGAAGCACCCCGGCAAGGGCCTGGAATACATCGGCTACATCAGCTACAACGGCATCACCTACCACAACCCCAGCCTGAAGTCCCGGATCACCATCAACCGGGACACCAGCAAGAACCAGTACTCCCTGCAGCTGAACAGCGTGACCCCCGAGGACACCGCCGTGTACTACTGCGCCCGGTACAGATACGACTACGACGGCGGCCACGCCATGGACTACTGGGGCCAGGGCACCCTGGTCACCGTGTCCTCT 45 OX40mAb17 VHQVQLQESGPGLVKPSQTLSLTCAVYGDSFSSGYWNWIRKHPGKGLEYIGYISYNGITYHNPSLKSRITINRDTSKNQFSLQLNSVTPEDTAVYYCARYRYDYDGGHAMDYWGQGTLVTVSS 46 OX40mAb VH17CAGGTGCAGCTGCAGGAAAGCGGCCCTGGCCTGGTCAAGCCCAGCCAGACCCTGAGCCTGAC DNACTGTGCCGTGTACGGCGACAGCTTCAGCAGCGGCTACTGGAACTGGATCCGGAAGCACCCCGGCAAGGGCCTGGAATACATCGGCTACATCAGCTACAACGGCATCACCTACCACAACCCCAGCCTGAAGTCCCGGATCACCATCAACCGGGACACCAGCAAGAACCAGTTCTCCCTGCAGCTGAACAGCGTGACCCCCGAGGACACCGCCGTGTACTACTGCGCCCGGTACAGATACGACTACGACGGCGGCCACGCCATGGACTACTGGGGCCAGGGCACCCTGGTCACCGTGTCCTCT 47 OX40mAb18 VHQVQLQESGPGLVKPSQTLSLTCAVYGGSFSSGYWNWIRKHPGKGLEYIGYISYNGITYHNPSLKSRITINPDTSKNQYSLQLNSVTPEDTAVYFCARYRYDYDGGHAMDYWGQGTLVTVSS 48 OX40mAb18 VHCAGGTGCAGCTGCAGGAAAGCGGCCCTGGCCTGGTCAAGCCCAGCCAGACCCTGAGCCTGAC DNACTGTGCCGTGTACGGCGGCAGCTTCAGCAGCGGCTACTGGAACTGGATCCGGAAGCACCCCGGCAAGGGCCTGGAATACATCGGCTACATCAGCTACAACGGCATCACCTACCACAACCCCAGCCTGAAGTCCCGGATCACCATCAACCCCGACACCAGCAAGAACCAGTACTCCCTGCAGCTGAACAGCGTGACCCCCGAGGACACCGCCGTGTACTTCTGCGCCCGGTACAGATACGACTACGACGGCGGCCACGCCATGGACTACTGGGGCCAGGGCACCCTGGTCACCGTGTCCTCT 49 OX40mAb19 VHQVQLQESGPGLVKPSQTLSLTCAVYGGSFSSGYWNWIRKHPGKGLEYIGYISYNGITYHNPSLKSRITINRDTSKNQYSLQLNSVTPEDTAVYFCARYRYDYDGGHAMDYWGQGTLVTVSS 50 OX40mAb19 VHCAGGTGCAGCTGCAGGAAAGCGGCCCTGGCCTGGTCAAGCCCAGCCAGACCCTGAGCCTGAC DNACTGTGCCGTGTACGGCGGCAGCTTCAGCAGCGGCTACTGGAACTGGATCCGGAAGCACCCCGGCAAGGGCCTGGAATACATCGGCTACATCAGCTACAACGGCATCACCTACCACAACCCCAGCCTGAAGTCCCGGATCACCATCAACCGGGACACCAGCAAGAACCAGTACTCCCTGCAGCTGAACAGCGTGACCCCCGAGGACACCGCCGTGTACTTCTGCGCCCGGTACAGATACGACTACGACGGCGGCCACGCCATGGACTACTGGGGCCAGGGCACCCTGGTCACCGTGTCCTCT 51 OX40mAb20 VHQVQLQESGPGLVKPSQTLSLTCAVYGGSFSSGYWNWIRKHPGKGLEYIGYISYNGITYHNPSLKSRITINRDTSKNQYSLQLNSVTPEDTAVYYCARYRYDYDGGHAMDYWGQGTLVTVSS 52 OX40mAb20 VHCAGGTGCAGCTGCAGGAAAGCGGCCCTGGCCTGGTCAAGCCCAGCCAGACCCTGAGCCTGAC DNACTGTGCCGTGTACGGCGGCAGCTTCAGCAGCGGCTACTGGAACTGGATCCGGAAGCACCCCGGCAAGGGCCTGGAATACATCGGCTACATCAGCTACAACGGCATCACCTACCACAACCCCAGCCTGAAGTCCCGGATCACCATCAACCGGGACACCAGCAAGAACCAGTACTCCCTGCAGCTGAACAGCGTGACCCCCGAGGACACCGCCGTGTACTACTGCGCCCGGTACAGATACGACTACGACGGCGGCCACGCCATGGACTACTGGGGCCAGGGCACCCTGGTCACCGTGTCCTCT 53 OX40mAb21 VHQVQLQESGPGLVKPSQTLSLTCAVYGGSFSSGYWNWIRKHPGKGLEYIGYISYNGITYHNPSLKSRITINPDTSKNQYSLQLNSVTPEDTAVYYCARYRYDYDGGHAMDYWGQGTLVTVSS 54 OX40mAb2l VHCAGGTGCAGCTGCAGGAAAGCGGCCCTGGCCTGGTCAAGCCCAGCCAGACCCTGAGCCTGAC DNACTGTGCCGTGTACGGCGGCAGCTTCAGCAGCGGCTACTGGAACTGGATCCGGAAGCACCCCGGCAAGGGCCTGGAATACATCGGCTACATCAGCTACAACGGCATCACCTACCACAACCCCAGCCTGAAGTCCCGGATCACCATCAACCCCGACACCAGCAAGAACCAGTACTCCCTGCAGCTGAACAGCGTGACCCCCGAGGACACCGCCGTGTACTACTGCGCCCGGTACAGATACGACTACGACGGCGGCCACGCCATGGACTACTGGGGCCAGGGCACCCTGGTCACCGTGTCCTCT 55 OX40mAb22 VHQVQLQESGPGLVKPSQTLSLTCAVYGGSFSSGYWNWIRKHPGKGLEYIGYISYNAITYHNPSLKSRITINRDTSKNQYSLQLNSVTPEDTAVYYCARYKYDYDAGHAMDYWGQGTLVTVSS 56 OX40mAb22 VHCAGGTGCAGCTGCAGGAAAGCGGCCCTGGCCTGGTCAAGCCCAGCCAGACCCTGAGCCTGAC DNACTGTGCCGTGTACGGCGGCAGCTTCAGCAGCGGCTACTGGAACTGGATCCGGAAGCACCCCGGCAAGGGCCTGGAATACATCGGCTACATCAGCTACAACGCCATCACCTACCACAACCCCAGCCTGAAGTCCCGGATCACCATCAACCGGGACACCAGCAAGAACCAGTACTCCCTGCAGCTGAACAGCGTGACCCCCGAGGACACCGCCGTGTACTACTGCGCCCGGTACAAATACGACTACGACGCCGGCCACGCCATGGACTACTGGGGCCAGGGCACCCTGGTCACCGTGTCCTCT 57 OX40mAb23 VHQVQLQESGPGLVKPSQTLSLTCAVYGGSFSSGYWNWIRKHPGKGLEYIGYISYNAITYHNPSLKSRITINRDTSKNQYSLQLNSVTPEDTAVYYCARYRYDYDGGHAMDYWGQGTLVTVSS 58 OX40mAb23 VHCAGGTGCAGCTGCAGGAAAGCGGCCCTGGCCTGGTCAAGCCCAGCCAGACCCTGAGCCTGAC DNACTGTGCCGTGTACGGCGGCAGCTTCAGCAGCGGCTACTGGAACTGGATCCGGAAGCACCCCGGCAAGGGCCTGGAATACATCGGCTACATCAGCTACAACGCCATCACCTACCACAACCCCAGCCTGAAGTCCCGGATCACCATCAACCGGGACACCAGCAAGAACCAGTACTCCCTGCAGCTGAACAGCGTGACCCCCGAGGACACCGCCGTGTACTACTGCGCCCGGTACAGATACGACTACGACGGCGGCCACGCCATGGACTACTGGGGCCAGGGCACCCTGGTCACCGTGTCCTCT 59 OX40mAb24 VHQVQLQESGPGLVKPSQTLSLTCAVYGGSFSSGYWNWIRKHPGKGLEYIGYISYNGITYHNPSLKSRITINRDTSKNQYSLQLNSVTPEDTAVYYCARYKYDYDGGHAMDYWGQGTLVTVSS 60 OX40mAb24 VHCAGGTGCAGCTGCAGGAAAGCGGCCCTGGCCTGGTCAAGCCCAGCCAGACCCTGAGCCTGAC DNACTGTGCCGTGTACGGCGGCAGCTTCAGCAGCGGCTACTGGAACTGGATCCGGAAGCACCCCGGCAAGGGCCTGGAATACATCGGCTACATCAGCTACAACGGCATCACCTACCACAACCCCAGCCTGAAGTCCCGGATCACCATCAACCGGGACACCAGCAAGAACCAGTACTCCCTGCAGCTGAACAGCGTGACCCCCGAGGACACCGCCGTGTACTACTGCGCCCGGTACAAATACGACTACGACGGCGGCCACGCCATGGACTACTGGGGCCAGGGCACCCTGGTCACCGTGTCCTCT 61 OX40mAb25 VHQVQLQESGPGLVKPSQTLSLTCAVYGGSFSSGYWNWIRKHPGKGLEYIGYISYSGITYHNPSLKSRITINRDTSKNQYSLQLNSVTPEDTAVYYCARYRYDYDGGHAMDYWGQGTLVTVSS 62 OX40mAb25 VHCAGGTGCAGCTGCAGGAAAGCGGCCCTGGCCTGGTCAAGCCCAGCCAGACCCTGAGCCTGAC DNACTGTGCCGTGTACGGCGGCAGCTTCAGCAGCGGCTACTGGAACTGGATCCGGAAGCACCCCGGCAAGGGCCTGGAATACATCGGCTACATCAGCTACAGCGGCATCACCTACCACAACCCCAGCCTGAAGTCCCGGATCACCATCAACCGGGACACCAGCAAGAACCAGTACTCCCTGCAGCTGAACAGCGTGACCCCCGAGGACACCGCCGTGTACTACTGCGCCCGGTACAGATACGACTACGACGGCGGCCACGCCATGGACTACTGGGGCCAGGGCACCCTGGTCACCGTGTCCTCT 63 OX40mAb25a VHQVQLQESGPGLVKPSQTLSLTCAVYGGSFSSGYWNWIRKHPGKGLEYIGYISYSGITYHNPSLKSRITINRDTSKNQYSLQLNSVTPEDTAVYYCARYKYDYDGGHAMDYWGQGTLVTVSS 64 OX40mAb25a VHCAGGTGCAGCTGCAGGAAAGCGGCCCTGGCCTGGTCAAGCCCAGCCAGACCCTGAGCCTGAC DNACTGTGCCGTGTACGGCGGCAGCTTCAGCAGCGGCTACTGGAACTGGATCCGGAAGCACCCCGGCAAGGGCCTGGAATACATCGGCTACATCAGCTACAGCGGCATCACCTACCACAACCCCAGCCTGAAGTCCCGGATCACCATCAACCGGGACACCAGCAAGAACCAGTACTCCCTGCAGCTGAACAGCGTGACCCCCGAGGACACCGCCGTGTACTACTGCGCCCGGTACAAATACGACTACGACGGCGGCCACGCCATGGACTACTGGGGCCAGGGCACCCTGGTCACCGTGTCCTCT 65 OX40mAb26 VHEVQLQESGPSLVKPSQTLSLTCSVTGDSFTSGYWNWIRKFPGNRLEYMGYISYNAITYHNPSLKSRISITRDTSKNHYYLQLNSVTTEDTATYFCARYRYDYDGGHAMDYWGQGTLVTVSS 66 OX40mAb26 VHGAGGTGCAGCTGCAGGAAAGCGGCCCCAGCCTGGTCAAGCCCAGCCAGACCCTGAGCCTGAC DNACTGCAGCGTGACCGGCGACAGCTTCACCAGCGGCTACTGGAACTGGATCCGGAAGTTCCCCGGCAACCGGCTCGAGTACATGGGCTACATCAGCTACAACGCCATCACCTACCACAACCCCAGCCTGAAGTCCCGGATCAGCATCACCCGGGACACCAGCAAGAACCACTACTACCTGCAGCTGAACAGCGTGACCACCGAGGACACCGCCACCTACTTTTGCGCCCGGTACAGATACGACTACGACGGCGGCCACGCCATGGACTACTGGGGCCAGGGCACCCTGGTCACCGTGTCCTCT 67 OX40mAb27 VHQVQLQESGPGLVKPSQTLSLTCAVYGGSFSSGYWNWIRKHPGKGLEYIGYISYNAITYHNPSLKSRITINRDTSKNQYSLQLNSVTPEDTAVYYCARYKYDYDGGHAMDYWGQGTLVTVSS 68 OX40mA27 VHCAGGTGCAGCTGCAGGAAAGCGGCCCTGGCCTGGTCAAGCCCAGCCAGACCCTGAGCCTGAC DNACTGTGCCGTGTACGGCGGCAGCTTCAGCAGCGGCTACTGGAACTGGATCCGGAAGCACCCCGGCAAGGGCCTGGAATACATCGGCTACATCAGCTACAACGCCATCACCTACCACAACCCCAGCCTGAAGTCCCGGATCACCATCAACCGGGACACCAGCAAGAACCAGTACTCCCTGCAGCTGAACAGCGTGACCCCCGAGGACACCGCCGTGTACTACTGCGCCCGGTACAAATACGACTACGACGGCGGCCACGCCATGGACTACTGGGGCCAGGGCACCCTGGTCACCGTGTCCTCT 69 HumanIgG1 CHASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVchainVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 70HumanIgG1 CHGCgTCgACCAAGGGCCCATCcGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCAchain DNACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCcTGGAACTCAGGCGCtCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTcTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCttaagCCTGTCTCCGGGTAAA 71 OX40mAb24 QVQLQESGPGLVKPSQTLSLTCAVYGGSFSSGYWNWIRKHPGKGLEYIGYISYNGITYHNPSLKSRITheavy INRDTSKNQYSLQLNSVTPEDTAVYYCARYKYDYDGGHAMDYWGQGTLVTVSSASTKGPSVFPLAchainPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 72 OX40mAb24 CAGGTGCAGCTGCAGGAAAGCGGCCCTGGCCTGGTCAAGCCCAGCCAGACCCTGAGCCTGAC heavyCTGTGCCGTGTACGGCGGCAGCTTCAGCAGCGGCTACTGGAACTGGATCCGGAAGCACCCCG chain DNAGCAAGGGCCTGGAATACATCGGCTACATCAGCTACAACGGCATCACCTACCACAACCCCAGCCTGAAGTCCCGGATCACCATCAACCGGGACACCAGCAAGAACCAGTACTCCCTGCAGCTGAACAGCGTGACCCCCGAGGACACCGCCGTGTACTACTGCGCCCGGTACAAATACGACTACGACGGCGGCCACGCCATGGACTACTGGGGCCAGGGCACCCTGGTCACCGTGTCCTCTGCgTCgACCAAGGGCCCATCcGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCcTGGAACTCAGGCGCtCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTcTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCttaagCCTGTCTCCGGGTAAA 73 OX40mAb28 QVQLQESGPGLVKPSQTLSLTCAVYGGSFSSGYWNWIRKHPGKGLEYIGYISYNGITYHNPSLKSRITheavy INRDTSKNQYSLQLNSVTPEDTAVYYCARYKYDYDGGHAMDYWGQGTLVTVSSASTKGPSVFPLAchainPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 74 OX40mAb28 CAGGTGCAGCTGCAGGAAAGCGGCCCTGGCCTGGTCAAGCCCAGCCAGACCCTGAGCCTGAC heavyCTGTGCCGTGTACGGCGGCAGCTTCAGCAGCGGCTACTGGAACTGGATCCGGAAGCACCCCG chain DNAGCAAGGGCCTGGAATACATCGGCTACATCAGCTACAACGGCATCACCTACCACAACCCCAGCCTGAAGTCCCGGATCACCATCAACCGGGACACCAGCAAGAACCAGTACTCCCTGCAGCTGAACAGCGTGACCCCCGAGGACACCGCCGTGTACTACTGCGCCCGGTACAAATACGACTACGACGGCGGCCACGCCATGGACTACTGGGGCCAGGGCACCCTGGTCACCGTGTCCTCTGCGTCGACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCTTGCAGCAGAAGCACCAGCGAGAGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGCGCTCTGACCAGCGGCGTGCATACCTTCCCCGCCGTGCTCCAGAGCAGCGGACTGTACTCCCTGAGCAGCGTGGTGACCGTGCCTTCCAGCAGCCTGGGCACCAAGACCTACACCTGCAACGTGGACCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTGGAGAGCAAGTACGGCCCTCCCTGCCCCCCTTGCCCTGCCCCCGAGTTCCTGGGCGGACCTAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGTCCCAGGAGGACCCCGAGGTCCAGTTTAATTGGTACGTGGACGGCGTGGAAGTGCATAACGCCAAGACCAAGCCCAGAGAGGAGCAGTTCAACAGCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTGCAAGGTCTCCAACAAGGGCCTGCCTAGCAGCATCGAGAAGACCATCAGCAAGGCCAAGGGCCAGCCACGGGAGCCCCAGGTCTACACCCTGCCACCTAGCCAAGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAAGGCTTCTATCCCAGCGATATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACTCCAGACTGACCGTGGACAAGTCCAGATGGCAGGAGGGCAACGTCTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCTGGGCAAG 75 OX40mAb29 QVQLQESGPGLVKPSQTLSLTCAVYGGSFSSGYWNWIRKHPGKGLEYIGYISYNGITYHNPSLKSRITheavy INRDTSKNQYSLQLNSVTPEDTAVYYCARYKYDYDGGHAMDYWGQGTLVTVSSASTKGPSVFPLAchainPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 76 OX40mAb29 CAGGTGCAGCTGCAGGAAAGCGGCCCTGGCCTGGTCAAGCCCAGCCAGACCCTGAGCCTGAC heavyCTGTGCCGTGTACGGCGGCAGCTTCAGCAGCGGCTACTGGAACTGGATCCGGAAGCACCCCG chain DNAGCAAGGGCCTGGAATACATCGGCTACATCAGCTACAACGGCATCACCTACCACAACCCCAGCCTGAAGTCCCGGATCACCATCAACCGGGACACCAGCAAGAACCAGTACTCCCTGCAGCTGAACAGCGTGACCCCCGAGGACACCGCCGTGTACTACTGCGCCCGGTACAAATACGACTACGACGGCGGCCACGCCATGGACTACTGGGGCCAGGGCACCCTGGTCACCGTGTCCTCTGCGTCGACCAAGGGCCCATCCGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCcTGGAACTCAGGCGCtCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATgcCCacCGTGCCCAGCACCTGAATTCGAGGGGGGAcCGTCAGTCTTCCTCTTCCCCCCAAAACCCaaGgACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTcTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 77 OX40mAb31 heavyQVQLQESGPGLVKPSQTLSLTCAVYGGSFSSGYWNWIRKHPGKGLEYIGYISYNAITYHNPSLKSRITchain INRDTSKNQYSLQLNSVTPEDTAVYYCARYKYDYDGGHAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 78 OX40mAb31 CAGGTGCAGCTGCAGGAAAGCGGCCCTGGCCTGGTCAAGCCCAGCCAGACCCTGAGCCTGAC heavyCTGTGCCGTGTACGGCGGCAGCTTCAGCAGCGGCTACTGGAACTGGATCCGGAAGCACCCCG chain DNAGCAAGGGCCTGGAATACATCGGCTACATCAGCTACAACGCCATCACCTACCACAACCCCAGCCTGAAGTCCCGGATCACCATCAACCGGGACACCAGCAAGAACCAGTACTCCCTGCAGCTGAACAGCGTGACCCCCGAGGACACCGCCGTGTACTACTGCGCCCGGTACAAATACGACTACGACGGCGGCCACGCCATGGACTACTGGGGCCAGGGCACCCTGGTCACCGTGTCCTCTGCGTCGACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCTTGCAGCAGAAGCACCAGCGAGAGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGCGCTCTGACCAGCGGCGTGCATACCTTCCCCGCCGTGCTCCAGAGCAGCGGACTGTACTCCCTGAGCAGCGTGGTGACCGTGCCTTCCAGCAGCCTGGGCACCAAGACCTACACCTGCAACGTGGACCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTGGAGAGCAAGTACGGCCCTCCCTGCCCCCCTTGCCCTGCCCCCGAGTTCCTGGGCGGACCTAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGTCCCAGGAGGACCCCGAGGTCCAGTTTAATTGGTACGTGGACGGCGTGGAAGTGCATAACGCCAAGACCAAGCCCAGAGAGGAGCAGTTCAACAGCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTGCAAGGTCTCCAACAAGGGCCTGCCTAGCAGCATCGAGAAGACCATCAGCAAGGCCAAGGGCCAGCCACGGGAGCCCCAGGTCTACACCCTGCCACCTAGCCAAGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAAGGCTTCTATCCCAGCGATATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACTCCAGACTGACCGTGGACAAGTCCAGATGGCAGGAGGGCAACGTCTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCTGGGCAAG 79 OX40mAb32 QVQLQESGPGLVKPSQTLSLTCAVYGGSFSSGYWNWIRKHPGKGLEYIGYISYNAITYHNPSLKSRITheavy INRDTSKNQYSLQLNSVTPEDTAVYYCARYKYDYDGGHAMDYWGQGTLVTVSSASTKGPSVFPLAchainPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 80 OX40mAb32 heavyCAGGTGCAGCTGCAGGAAAGCGGCCCTGGCCTGGTCAAGCCCAGCCAGACCCTGAGCCTGAC chain DNACTGTGCCGTGTACGGCGGCAGCTTCAGCAGCGGCTACTGGAACTGGATCCGGAAGCACCCCGGCAAGGGCCTGGAATACATCGGCTACATCAGCTACAACGCCATCACCTACCACAACCCCAGCCTGAAGTCCCGGATCACCATCAACCGGGACACCAGCAAGAACCAGTACTCCCTGCAGCTGAACAGCGTGACCCCCGAGGACACCGCCGTGTACTACTGCGCCCGGTACAAATACGACTACGACGGCGGCCACGCCATGGACTACTGGGGCCAGGGCACCCTGGTCACCGTGTCCTCTGCGTCGACCAAGGGCCCATCCGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCcTGGAACTCAGGCGCtCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATgcCCacCGTGCCCAGCACCTGAATTCGAGGGGGGAcCGTCAGTCTTCCTCTTCCCCCCAAAACCCaaGgACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTcTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 81 OX40mAb37 heavyQVQLQESGPGLVKPSQTLSLTCAVYGGSFSSGYWNWIRKHPGKGLEYIGYISYNGITYHNPSLKSRITchain INRDTSKNQYSLQLNSVTPEDTAVYYCARYKYDYDGGHAMDYWGQGTLVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK 82 OX40mAb37 heavyCAGGTGCAGCTGCAGGAAAGCGGCCCTGGCCTGGTCAAGCCCAGCCAGACCCTGAGCCTGAC chain DNACTGTGCCGTGTACGGCGGCAGCTTCAGCAGCGGCTACTGGAACTGGATCCGGAAGCACCCCGGCAAGGGCCTGGAATACATCGGCTACATCAGCTACAACGGCATCACCTACCACAACCCCAGCCTGAAGTCCCGGATCACCATCAACCGGGACACCAGCAAGAACCAGTACTCCCTGCAGCTGAACAGCGTGACCCCCGAGGACACCGCCGTGTACTACTGCGCCCGGTACAAATACGACTACGACGGCGGCCACGCCATGGACTACTGGGGCCAGGGCACCCTGGTCACCGTGTCCTCTGCGaaGACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGTGGTTGTAAGCCTTGCATATGTACcGTCCCAGAAGTATCATCTGTCTTCATCTTCCCCCCAAAGCCCAAGGATGTGCTCACCATTACTCTGACTCCTAAGGTCACGTGTGTTGTGGTAGACATCAGCAAGGATGATCCCGAGGTCCAGTTCAGCTGGTTTGTAGATGATGTGGAGGTGCACACAGCTCAGACGCAACCCCGGGAGGAGCAGTTCAACAGCACTTTCCGCTCAGTCAGTGAACTTCCCATCATGCACCAGGACTGGCTCAATGGCAAGGAGTTCAAATGCAGGGTCAACAGTGCAGCTTTCCCTGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGCAGACCGAAGGCTCCACAGGTGTAtACCATTCCACCTCCCAAGGAGCAGATGGCCAAGGATAAAGTCAGTCTGACCTGCATGATAACAGACTTCTTCCCTGAAGACATTACTGTGGAGTGGCAGTGGAATGGGCAGCCAGCGGAGAACTACAAGAACACTCAGCCCATCATGGACACAGATGGCTCTTACTTCGTCTACAGCAAGCTCAATGTGCAGAAGAGCAACTGGGAGGCAGGAAATACTTTCACCTGCTCTGTGTTACATGAGGGCCTGCACAACCACCATACTGAGAAGAGCCTCTCCCACTCTCCTGG TAAA 83OX40mAb37 lightDIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSKLHSGVPSRFSGSGSchainGTDYTLTISSLQPEDFATYYCQQGSALPWTFGQGTKVEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC 84 OX40mAb37 lightGACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACAGAGTGACCAT chain DNACACCTGTCGGGCCAGCCAGGACATCAGCAACTACCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACTACACCAGCAAGCTGCACAGCGGCGTGCCCAGCAGATTCAGCGGCAGCGGCTCCGGCACCGACTACACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGGCTCCGCCCTGCCCTGGACCTTTGGCCAGGGCACCAAGGTGGAAATCAAGCGGGCTGATGCGGCGCCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGT 85 OX86 VHQVQLKESGPGLVQPSQTLSLTCTVSGFSLTGYNLHWVRQPPGKGLEWMGRMRYDGDTYYNSVLKSRLSISRDTSKNQVFLKMNSLQTDDTAIYYCTRDGRGDSFDYWGQGVMVTVSS 86 OX86 heavy QVQLKESGPGLVQPSQTLSLTCTVSGFSLTGYNLHWVRQPPGKGLEWMGRMRYDGDIYYNSVLK chainSRLSISRDTSKNQVFLKMNSLQTDDTAIYYCTRDGRGDSFDYWGQGVMVTVSSASTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK 87 OX86 heavy CAGGTGCAGCTGAAGGAGTCAGGACCTGGTCTGGTGCAGCCCTCACAGACCCTGTCCCTCACCchain DNATGCACTGTCTCTGGGTTCTCACTAACCGGTTACAATTTACACTGGGTTCGCCAGCCTCCAGGAAAGGGTCTGGAGTGGATGGGAAGAATGAGGTATGATGGAGACACATATTATAATTCAGTTCTCAAATCCCGACTGAGCATCAGCAGGGACACCTCCAAGAACCAAGTTTTCTTGAAAATGAACAGTCTGCAAACGGATGACACAGCCATTTACTATTGTACCAGAGACGGGCGTGGTGACTCCTTTGATTACTGGGGCCAAGGAGTCATGGTCACAGTCTCCTCCGCGTCGACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGTGGTTGTAAGCCTTGCATATGTACCGTCCCAGAAGTATCATCTGTCTTCATCTTCCCCCCAAAGCCCAAGGATGTGCTCACCATTACTCTGACTCCTAAGGTCACGTGTGTTGTGGTAGACATCAGCAAGGATGATCCCGAGGTCCAGTTCAGCTGGTTTGTAGATGATGTGGAGGTGCACACAGCTCAGACGCAACCCCGGGAGGAGCAGTTCAACAGCACTTTCCGCTCAGTCAGTGAACTTCCCATCATGCACCAGGACTGGCTCAATGGCAAGGAGTTCAAATGCAGGGTCAACAGTGCAGCTTTCCCTGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGCAGACCGAAGGCTCCACAGGTGTATACCATTCCACCTCCCAAGGAGCAGATGGCCAAGGATAAAGTCAGTCTGACCTGCATGATAACAGACTTCTTCCCTGAAGACATTACTGTGGAGTGGCAGTGGAATGGGCAGCCAGCGGAGAACTACAAGAACACTCAGCCCATCATGGACACAGATGGCTCTTACTTCGTCTACAGCAAGCTCAATGTGCAGAAGAGCAACTGGGAGGCAGGAAATACTTTCACCTGCTCTGTGTTACATGAGGGCCTGCACAACCACCATACTGAGAAGAGCCTCTCCCACTCTCCTGGTAAA 88 OX86 VLDIVMTQGALPNPVPSGESASITCRSSQSLVYKDGQTYLNWFLQRPGQSPQLLTYWMSTRASGVSDRFSGSGSGTYFTLKISRVRAEDAGVYYCQQVREYPFTFGSGTKLEIK 89 OX86 light DIVMTQGALPNPVPSGESASITCRSSQSLVYKDGQTYLNWFLQRPGQSPQLLTYWMSTRASGVSD chainRFSGSGSGTYFTLKISRVRAEDAGVYYCQQVREYPFTFGSGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC 90 OX86 light GATATTGTGATGACCCAGGGTGCACTCCCCAATCCTGTCCCTTCTGGAGAGTCAGCTTCCATCA chainCCTGCAGGTCTAGTCAGAGTCTGGTATACAAAGACGGCCAGACATACTTGAATTGGTTTCTGCA DNAGAGGCCAGGACAGTCTCCTCAGCTTCTGACCTATTGGATGTCTACCCGTGCATCAGGAGTCTCAGACAGGTTCAGTGGCAGTGGGTCAGGAACATATTTCACACTGAAAATCAGTAGAGTGAGGGCTGAGGATGCGGGTGTGTATTACTGTCAGCAAGTTCGAGAGTATCCTTTCACTTTCGGCTCAGGGACGAAGTTGGAAATAAAACGGGCTGATGCGGCGCCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGT 91 Human OX40MCVGARRLGRGPCAALLLLGLGLSTVTGLHCVGDTYPSNDRCCHECRPGNGMVSRCSRSQNTVCRPCGPGFYNDVVSSKPCKPCTWCNLRSGSERKQLCTATQDTVCRCRAGTQPLDSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGKHTLQPASNSSDAICEDRDPPATQPQETQGPPARPITVQPTEAWPRTSQGPSTRPVEVPGGRAVAAILGLGLVLGLLGPLAILLALYLLRRDQRLPPDHKPPGGGSFRTPIQEEQADAHSTLAKI 92 Mouse OX40MYVWVQQPTALLLLGLTLGVTARRLNCVKHTYPSGHKCCRECQPGHGMVSRCDHTRDTLCHPCETGFYNEAVNYDTCKQCTQCNHRSGSELKQNCTPTQDTVCRCRPGTQPRQDSGYKLGVDCVPCPPGHFSPGNNQACKPWTNCTLSGKQTRHPASDSLDAVCEDRSLLATLLWETQRPTFRPTTVQSTTVWPRTSELPSPPTLVTPEGPAFAVLLGLGLGLLAPLTVLLALYLLRKAWRLPNTPKPCWGNSFRTPIQEEHTDAHFTLAKI

What is claimed is:
 1. An isolated antibody or fragment thereofcomprising a humanized heavy chain variable region (VH) and a humanizedlight chain variable region (VL); wherein the VH comprises: (a) a CDR1comprising the amino acid sequence of SEQ ID NO: 8, (b) a CDR2comprising the amino acid sequence of SEQ ID NO: 14, and (c) a CDR1comprising the amino acid sequence of SEQ ID NO: 27, and wherein the VLcomprises: (d) a CDR1 comprising the amino acid sequence of residues24-34 of SEQ ID NO: 29, (e) a CDR2 comprising the amino acid sequence ofresidues 50-56 of SEQ ID NO: 29, and (f) a CDR3 comprising the aminoacid sequence of residues 89-97 of SEQ ID NO: 29, wherein the antibodyor fragment thereof can specifically bind to human OX40.
 2. The antibodyor fragment of claim 1, wherein the antigen-binding fragment is an Fvfragment, an Fab fragment, an F(ab')2 fragment, an Fab' fragment, a dsFvfragment, an scFv fragment, or an sc(Fv)2 fragment, or any combinationthereof.
 3. The antibody or fragment thereof of claim 1, which caninduce dose-dependent proliferation of activated CD4⁺ T cells anddose-dependent cytokine release in primary activated human CD4⁺ T cellsin a plate-based assay.
 4. The antibody or fragment thereof of claim 3,wherein a 20% maximal proliferation response (EC₂₀) can be achieved inprimary activated human CD4⁺ T cells at an antibody concentration ofabout 14 pM to about 28 pM, a 50% maximal proliferation response (EC₅₀)can be achieved in primary activated human CD4⁺ T cells at an antibodyconcentration of about 0.3 pM to about 130 pM, and a 90% maximalproliferation response (EC₉₀) can be achieved in primary activated humanCD4⁺ T cells at an antibody concentration of about 50 pM to about 90 pM,all as measured by flow cytometry.
 5. The antibody or fragment thereofof claim 4, wherein EC₂₀ is about 21 pM, EC₅₀ is about 28 pM, and EC₉₀is about 72 pM.
 6. The antibody or fragment thereof of claim 3, whereinthe cytokine is IFNγ, TNFα, IL-5, IL-10, IL-2, IL-4, IL-13, IL-8, IL-12p70, IL-1β, or any combination thereof.
 7. The antibody or fragmentthereof of claim 6 wherein the cytokine is IFNγ, TNFα, IL-5, IL-10,IL-13, or any combination thereof.
 8. The antibody or fragment thereofof claim 1, which can achieve CD4⁺ T cell proliferation and cytokinerelease in primary activated cynomolgus monkey CD4⁺ T cells and inprimary activated rhesus monkey CD4⁺ T cells.
 9. The antibody orfragment thereof of claim 1, which can activate the NFκB pathway in OX40expressing T cells in the presence of FcγR-expressing cells.
 10. Theantibody or fragment thereof of claim 9, wherein the OX40-expressing Tcells are OX40-expressing Jurkat NFκB-luciferase reporter cells thatproduce luciferase in response to stimulation of the NFκB signalingpathway.
 11. The antibody or fragment thereof of claim 1, which cantrigger complement-dependent or antibody-dependent cellular cytotoxicityagainst OX40-expressing cells.
 12. The antibody or fragment thereof ofclaim 11, which can bind to C1q and trigger NK-mediatedantibody-dependent cellular cytotoxicity against the OX40-expressingcells.
 13. The antibody or fragment thereof of claim 1, whereinadministration of an effective dose to a subject in need of cancertreatment can inhibit tumor growth in the subject.
 14. The antibody orfragment thereof of claim 13, wherein the tumor growth inhibition isachieved in the presence of T cells.
 15. The antibody or fragmentthereof of claim 13, wherein tumor growth is inhibited by at least 10%,at least 20%, at least 30%, at least 40%, and least 50%, at least 60%,or at least 70% compared to administration of an isotype-matched controlantibody or fragment thereof.
 16. A composition comprising the antibodyor fragment thereof of claim 1, and a carrier.
 17. An antibody orantigen-binding fragment thereof comprising a humanized heavy chainvariable region (VH) and a humanized light chain variable region (VL),wherein the VH comprises the amino acid sequence SEQ ID NO: 59, whereinthe VL comprises the amino acid sequence SEQ ID NO: 29, and wherein theantibody or fragment thereof can specifically bind to human OX40. 18.The antibody or fragment thereof of claim 17 comprising the heavy chainamino acid sequence SEQ ID NO: 71 and the light chain amino acidsequence SEQ ID NO:
 30. 19. A polynucleotide comprising a nucleic acidthat encodes the antibody or fragment thereof, of claim
 17. 20. Thepolynucleotide of claim 19, comprising the nucleic acid of SEQ ID NO:60, the nucleic acid of SEQ ID NO: 31, the nucleic acid of SEQ ID NO:72, or any combination thereof.
 21. A vector comprising thepolynucleotide of claim
 19. 22. A host cell comprising thepolynucleotide of claim
 19. 23. A method of producing an antibody orfragment thereof, comprising culturing the host cell of claim 22 underconditions in which the antibody or fragment thereof encoded by thepolynucleotide is expressed, and recovering the antibody or fragmentthereof.
 24. A host cell comprising the vector of claim 21.